Methods for making ultrasound contrast agents

ABSTRACT

Provided herein are improved methods for preparing phospholipid formulations including phospholipid UCA formulations.

BACKGROUND

Lipid-encapsulated gas microspheres are used as contrast agents inultrasound imaging applications.

SUMMARY

The disclosure provides improved methods for preparingphospholipid-based ultrasound contrast agents. The disclosure is basedin part on the unexpected finding that certain phospholipid-basedultrasound contrast agent formulations are susceptible to divalent metalcations. In the presence of particular levels of certain of thosecations, phospholipids and potentially other components in theformulation precipitate rendering the formulation unusable. It was notheretofore appreciated that certain divalent metal cations had theability to so negatively impact the ultrasound contrast agentformulation.

Based on these findings, this disclosure contemplates improved methodsfor synthesizing such formulations that prevent such unwantedphospholipid precipitation as well as the formulations resulting fromsuch methods. Also provided are methods for using these improvedformulations in the synthesis of improved ultrasound contrast agents,and their use in imaging subjects in need thereof.

Thus, in one aspect, provided herein is a method for preparing aphospholipid suspension, comprising providing DPPA, DPPC andMPEG5000-DPPE stocks, measuring calcium concentration of one or more orall of the DPPC, DPPA and MPEG5000-DPPE stocks, combining the DPPA, DPPCand/or MPEG5000-DPPE stocks with a non-aqueous solvent to form aphospholipid solution, and combining the phospholipid solution with anaqueous solvent to form a phospholipid suspension. In some embodiments,the method further comprises measuring calcium concentration of thenon-aqueous solvent. In some embodiments, the combined measured calciumconcentration of the DPPA. DPPC and/or MPEG5000-DPPE stocks is low(i.e., the sum of or the combined calcium concentration for the measuredstocks, whether such measured stocks are DPPA alone, DPPC alone,MPEG5000-DPPE alone, DPPA and DPPC, DPPA and MPEG5000-DPPE, DPPC, andMPEG5000-DPPE, or DPPA, DPPC and MPEG5000-DPPE, is low).

In some embodiments, the combined measured calcium concentration of theDPPA. DPPC and/or MPEG-DPPE stocks and the non-aqueous solvent is low(i.e., the sum of or the combined calcium concentration for the measuredcomponents, whether such measured components are DPPA alone or DPPA andnon-aqueous solvent, DPPC alone or DPPA and non-aqueous solvent,MPEG5000-DPPE alone or MPEG5000-DPPE and non-aqueous solvent, DPPA andDPPC or DPPA, DPPC and non-aqueous solvent, DPPA and MPEG5000-DPPE orDPPA, MPEG5000-DPPE and non-aqueous solvent, DPPC and MPEG5000-DPPE orDPPC. MPEG5000-DPPE and non-aqueous solvent, DPPA, DPPC andMPEG5000-DPPE or DPPA, DPPC, MPEG5000-DPPE and non-aqueous solvent, islow).

In some embodiments, the calcium concentrations of the DPPC, DPPA andMPEG5000-DPPE stocks are measured.

In some embodiments, the calcium concentrations of the DPPC, DPPA andMPEG5000-DPPE stocks are measured and the combined measured calciumconcentration of the DPPA, DPPC, MPEG-DPPE stocks and the non-aqueoussolvent is low.

Thus, depending on the embodiment, only the calcium concentration ofDPPA is measured, or only the calcium concentration of DPPC is measured,or only the calcium concentration of MPEG5000-DPPE is measured, or onlythe calcium concentrations of DPPA and DPPC are measured (and suchconcentrations are added together to yield a combined measured calciumconcentration), or only the calcium concentrations of DPPA andMPEG5000-DPPE are measured (and such concentrations are added togetherto yield a combined measured calcium concentration), or only the calciumconcentrations of DPPC and MPEG5000-DPPE are measured (and suchconcentrations are added together to yield a combined measured calciumconcentration), or only the calcium concentrations of DPPA, DPPC andMPEG5000-DPPE are measured (and such concentrations are added togetherto yield a combined measured calcium concentration), or only the calciumconcentrations of DPPA and non-aqueous solvent are measured (and suchconcentrations are added together to yield a combined measured calciumconcentration), or only the calcium concentrations of DPPC. andnon-aqueous solvent are measured (and such concentrations are addedtogether to yield a combined measured calcium concentration), or onlythe calcium concentrations of MPEG5000-DPPE and non-aqueous solvent aremeasured (and such concentrations are added together to yield a combinedmeasured calcium concentration), or only the calcium concentrations ofDPPA, DPPC and non-aqueous solvent are measured (and such concentrationsare added together to yield a combined measured calcium concentration),or only the calcium concentrations of DPPA, MPEG5000-DPPE andnon-aqueous solvent are measured (and such concentrations are addedtogether to yield a combined measured calcium concentration), or onlythe calcium concentrations of DPPC, MPEG5000-DPPE and non-aqueoussolvent are measured (and such concentrations are added together toyield a combined measured calcium concentration), or the calciumconcentrations of DPPA, DPPC, MPEG5000-DPPE and non-aqueous solvent aremeasured (and such concentrations are added together to yield a combinedmeasured calcium concentration). It should be clear that everycombination of components is contemplated in arriving at a combinedmeasured or characterized (as discussed below) calcium concentration. Itis to be understood that the terms DPPA, DPPA lipid, DPPA phospholipid,DPPA stock, DPPA lipid stock, and DPPA phospholipid stock are usedinterchangeably unless explicitly stated otherwise. Similarinterchangeable terms are used for DPPC and MPEG5000-DPPE.

In another aspect, a variation of the foregoing method is provided. Suchmethod comprises providing DPPC and MPEG5000-DPPE stocks, measuringcalcium concentration of one or both of the DPPC and MPEG-5000-DPPEstocks, combining the DPPC and MPEG-5000-DPPE stocks with a non-aqueoussolvent to form a phospholipid solution, and combining the phospholipidsolution with an aqueous solvent to form a phospholipid suspension. Insome embodiments, the method further comprises measuring calciumconcentration of the non-aqueous solvent. Various of the foregoingembodiments apply equally to this method and should be so understood.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising providing DPPA, DPPC andMPEG5000-DPPE, stocks, measuring calcium concentration of one or more orall of the DPPC, DPPA and MPEG5000-DPPE stocks, combining DPPA, DPPCand/or MPEG5000-DPPE stocks having a combined measured low calciumconcentration with a non-aqueous solvent to form a phospholipidsolution, and combining the phospholipid solution with an aqueoussolvent to form a phospholipid suspension. In some embodiments, themethod further comprises measuring the calcium concentration of thenon-aqueous solvent and the DPPA, MPEG500-DPPE stocks and thenon-aqueous solvent have a combined measured low calcium concentration.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising combining aMPEG5000-DPPE stock, a DPPA stock, a DPPC stock and a non-aqueoussolvent, each with characterized calcium concentration to form aphospholipid solution, wherein the combined characterized calciumconcentration of the MPEG5000-DPPE stock, the DPPA stock, the DPPC stockand the non-aqueous solvent is a low calcium concentration, andcombining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising combining aMPEG5000-DPPE stock, a DPPC stock and a non-aqueous solvent, each withcharacterized calcium concentration to form a phospholipid solution,wherein the combined characterized calcium concentration of theMPEG5000-DPPE stock, the DPPC stock and the non-aqueous solvent is a lowcalcium concentration, and combining the phospholipid solution with anaqueous solvent to form a phospholipid suspension.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising selecting aMPEG5000-DPPE stock, a DPPA stock and a DPPC stock, one, two or allthree of which have a characterized calcium concentration, wherein thecombined characterized calcium concentration is a low calciumconcentration, combining said MPEG5000-DPPE stock, DPPA stock, DPPCstock and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form aphospholipid suspension. Components such as phospholipid stocks and/ornon-aqueous solvent are so selected on the basis of having an individualor a combined characterized calcium concentration.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising selecting aMPEG5000-DPPE stock and a DPPC stock, one or both of which have acharacterized calcium concentration, wherein the combined characterizedcalcium concentration is a low calcium concentration, combining saidMPEG5000-DPPE stock, DPPC stock and a non-aqueous solvent to form aphospholipid solution, and combining the phospholipid solution with anaqueous solvent to form a phospholipid suspension. Components such asphospholipid stocks and/or non-aqueous solvent are so selected on thebasis of having an individual or a combined characterized calciumconcentration.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising selecting aMPEG5000-DPPE stock, a DPPA stock and a DPPC stock, each withcharacterized calcium concentration, wherein the combined characterizedcalcium concentration is a low calcium concentration, combining saidMPEG5000-DPPE stock, DPPA stock. DPPC stock and a non-aqueous solvent toform a phospholipid solution, and combining the phospholipid solutionwith an aqueous solvent to form a phospholipid suspension. In someembodiments, the non-aqueous solvent has a characterized calciumconcentration, and the combined characterized calcium concentration ofthe MPEG5000-DPPE, DPPA and DPPC stocks and the non-aqueous solvent islow.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising measuring calciumconcentration of a MPEG5000-DPPE stock, combining a MPEG5000-DPPE stockhaving a measured low calcium concentration with a DPPA stock, a DPPCstock, and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form aphospholipid suspension. In some embodiments, the low calciumconcentration is less than 115 ppm,

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising measuring calciumconcentration of a MPEG5000-DPPE stock, combining a MPEG5000-DPPE stockhaving a measured low calcium concentration with a DPPC stock, and anon-aqueous solvent to form a phospholipid solution, and combining thephospholipid solution with an aqueous solvent to form a phospholipidsuspension. In some embodiments, the low calcium concentration is lessthan 115 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension comprising measuring calciumconcentration of a DPPC stock, combining a DPPC stock having a measuredlow calcium concentration with a DPPA stock, a MPEG5000-DPPE stock, anda non-aqueous solvent to form a phospholipid solution, and combining thephospholipid solution with an aqueous solvent to form a phospholipidsuspension. In some embodiments, the low calcium concentration is lessthan 90 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension comprising measuring calciumconcentration of a DPPC stock, combining a DPPC stock having a measuredlow calcium concentration with a MPEG5000-DPPE stock, and a non-aqueoussolvent to form a phospholipid solution, and combining the phospholipidsolution with an aqueous solvent to form a phospholipid suspension. Insome embodiments, the low calcium concentration is less than 90 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension comprising measuring calciumconcentration of a DPPA stock, combining a DPPA stock having a measuredlow calcium concentration with a DPPC stock, a MPEG5000-DPPE stock, anda non-aqueous solvent to form a phospholipid solution, and combining thephospholipid solution with an aqueous solvent to form a phospholipidsuspension. In some embodiments, the low calcium concentration is lessthan 780 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension comprising measuring calciumconcentration of a non-aqueous solvent, combining a non-aqueous solventhaving a measured low calcium concentration with a DPPA stock, a DPPCstock, and a MPEG5000-DPPE stock, to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form aphospholipid suspension. In some embodiments, the low calciumconcentration is less than 0.7 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension comprising combining MPEG5000-DPPE,DPPA and DPPC stocks with a non-aqueous solvent to form a phospholipidsolution, measuring calcium concentration of the phospholipid solution,and combining a phospholipid solution having a measured low calciumconcentration with an aqueous solvent to form a phospholipid suspension.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising selecting aMPEG5000-DPPE stock characterized as having no or low calciumconcentration, combining said MPEG5000-DPPE stock, a DPPA stock, a DPPCstock and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form aphospholipid suspension. In some embodiments, the MPEG5000-DPPE stock isfurther characterized as having no or low divalent metal cation content.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising combining aMPEG5000-DPPE stock, a DPPA stock, a DPPC stock and a non-aqueoussolvent to form a phospholipid solution characterized as having no orlow calcium concentration, and combining the phospholipid solution withan aqueous solvent to form a phospholipid suspension.

This disclosure further provides in another aspect a method for imaginga subject comprising combining a phospholipid suspension with aperfluorocarbon gas to form an ultrasound contrast agent comprisingphospholipid-encapsulated gas microspheres, administering the ultrasoundcontrast agent to a subject, and obtaining one or more contrast-enhancedultrasound contrast images of the subject, wherein the phospholipidsuspension is prepared by any of the foregoing methods.

This disclosure further provides in another aspect a method for imaginga subject comprising combining a phospholipid suspension with aperfluorocarbon gas to form an ultrasound contrast agent comprisingphospholipid-encapsulated gas microspheres, administering the ultrasoundcontrast agent to a subject, and obtaining one or more contrast-enhancedultrasound contrast images of the subject, wherein the phospholipidsuspension is prepared by a method comprising measuring calciumconcentration of MPEG5000-DPPE stock, combining a MPEG5000-DPPE stockhaving a measured low calcium concentration with a DPPA stock, a DPPCstock, and a non-aqueous solvent to form a phospholipid solution, andcombining the phospholipid solution with an aqueous solvent to form thephospholipid suspension.

This disclosure further provides in another aspect a method for imaginga subject comprising combining a phospholipid suspension with aperfluorocarbon gas to form an ultrasound contrast agent comprisingphospholipid-encapsulated gas microspheres, administering the ultrasoundcontrast agent to a subject, and obtaining one or more contrast-enhancedultrasound contrast images of the subject, wherein the phospholipidsuspension is prepared by a method comprising combining MPEG5000-DPPE,DPPA and DPPC stocks with a non-aqueous solvent to form a phospholipidsolution, measuring calcium concentration of the phospholipid solution,and combining a phospholipid solution having a measured low calciumconcentration with an aqueous solvent to form a phospholipid suspension.

This disclosure further provides in another aspect a method for imaginga subject comprising combining a phospholipid suspension with aperfluorocarbon gas to form an ultrasound contrast agent comprisingphospholipid-encapsulated gas microspheres, administering the ultrasoundcontrast agent to a subject, and obtaining one or more contrast-enhancedultrasound contrast images of the subject, wherein the phospholipidsuspension is prepared by a method comprising selecting a MPEG5000-DPPEstock characterized as having no or low calcium concentration, combiningsaid MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and a non-aqueoussolvent to form a phospholipid solution, and combining the phospholipidsolution with an aqueous solvent to form a phospholipid suspension.

This disclosure further provides in another aspect a method for imaginga subject comprising combining a phospholipid suspension with aperfluorocarbon gas to form an ultrasound contrast agent comprisingphospholipid-encapsulated gas microspheres, administering the ultrasoundcontrast agent to a subject, and obtaining one or more contrast-enhancedultrasound contrast images of the subject, wherein the phospholipidsuspension is prepared by a method comprising combining a MPEG5000-DPPEstock, a DPPA stock, a DPPC stock and a non-aqueous solvent to form aphospholipid solution characterized as having no or low calciumconcentration, and combining the phospholipid solution with an aqueoussolvent to form a phospholipid suspension.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising individually combiningDPPA, DPPC and MPEG5000-DPPE stocks with a PG-comprising non-aqueoussolvent, in a low or no calcium condition, to form a phospholipidsolution, and combining the phospholipid solution with an aqueoussolvent to form a phospholipid suspension. In some embodiments, the noor low calcium condition is less than 0.7 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising sequentially combiningDPPA, DPPC and MPEG5000-DPPE stocks with a PG-comprising non-aqueoussolvent, in a low or no calcium condition, in an order-independentmanner, to form a phospholipid solution, and combining the phospholipidsolution with an aqueous solvent to form a phospholipid suspension. Insome embodiments, the no or low calcium condition is less than 0.7 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising combining, in a methanoland toluene-free condition, DPPA, DPPC and MPEG5000-DPPE stocks to forma phospholipid blend, combining the phospholipid blend with aPG-comprising non-aqueous solvent, in a low or no calcium condition, toform a phospholipid solution, and combining the phospholipid solutionwith an aqueous solvent to form a phospholipid suspension. In someembodiments, the no or low calcium condition is less than 0.7 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising combining DPPA, DPPC andMPEG5000-DPPE stocks with a blend solvent to form a phospholipid blend,evaporating the blend solvent to form a dried phospholipid blend,combining the dried phospholipid blend with a PG-comprising non-aqueoussolvent, in a low or no calcium condition, to form a phospholipidsolution, and combining the phospholipid solution with an aqueoussolvent to form a phospholipid suspension. In some embodiments, the noor low calcium condition is less than 0.7 ppm.

This disclosure further provides in another aspect a method forpreparing a phospholipid suspension, comprising combining DPPA, DPPC andMPEG5000-DPPE stocks with a blend solvent to form a phospholipid blend,precipitating, in a MTBE-free condition, the phospholipid blend using asecond blend solvent, combining the precipitated phospholipid blend witha non-aqueous solvent, in a low or no calcium condition, to form aphospholipid solution, and combining the phospholipid solution with anaqueous solvent to form a phospholipid suspension. In some embodiments,the no or low calcium condition is less than 0.7 ppm,

In some embodiments of any of the foregoing methods, the method furthercomprises combining the phospholipid suspension with a perfluorocarbongas to form an ultrasound contrast agent comprisingphospholipid-encapsulated gas microspheres. In some embodiments of anyof the foregoing methods, the method further comprises administering theultrasound contrast agent to a subject and obtaining one or morecontrast-enhanced ultrasound images of the subject.

This disclosure further provides in another aspect a method for imaginga subject comprising combining a phospholipid suspension with aperfluorocarbon gas to form an ultrasound contrast agent comprisingphospholipid-encapsulated gas microspheres, administering the ultrasoundcontrast agent to a subject, and obtaining one or more contrast-enhancedultrasound contrast images of the subject, wherein the phospholipidsuspension is prepared by any one of the foregoing methods.

This disclosure further provides in other aspects a compositioncomprising a phospholipid solution comprising DPPA, DPPC andMPEG5000-DPPE in a non-aqueous solvent and having a low calciumconcentration, as well as a composition comprising a phospholipidsolution comprising DPPA, DPPC and MPEG5000-DPPE in a non-aqueoussolvent, wherein the DPPA, DPPC and MPEG5000-DPPE and the non-aqueoussolvent have a combined characterized calcium ion content that is low.In some embodiments, the non-aqueous solvent comprises propylene glycol(e.g., propylene glycol may be the only non-aqueous solvent or it may beused in combination with one or more other solvents to render anon-aqueous solvent). In some embodiments, the non-aqueous solventcomprises propylene glycol and glycerol. In some embodiments, thenon-aqueous solvent comprises a buffer. In some embodiments, the bufferis acetate buffer. In some embodiments, the composition comprises aperfluorocarbon gas. In some embodiments, the perfluorocarbon gas isperflutren. Thus, in some instances, the composition is provided in acontainer such as a vial, and the gas is provided in the headspace ofthe container. Also provided are methods for combining the phospholipidsolution with the perfluorocarbon gas to form an ultrasound contrastagent comprising phospholipid-encapsulated gas microspheres. The methodmay further comprise administering the ultrasound contrast agent to asubject and Obtaining one or more contrast-enhanced ultrasound images ofthe subject.

In some embodiments of the various aspects provided herein, thenon-aqueous solvent comprises (i) propylene glycol or (ii) propyleneglycol and glycerol.

In some embodiments of the various aspects provided herein, thenon-aqueous solvent comprises a buffer. In some embodiments of thevarious aspects provided herein, the non-aqueous solvent comprises anacetate buffer.

In some embodiments of the various aspects provided herein, the aqueoussolvent comprises a buffer. In some embodiments of the various aspectsprovided herein, the aqueous solvent comprises a phosphate buffer.

In some embodiments of the various aspects provided herein, the DPPC,DPPA and MPEG5000-DPPE stocks are individually combined with thenon-aqueous solvent to form the phospholipid solution.

In some embodiments of the various aspects provided herein, the DPPC,DPPA and MPEG5000-DPPE stocks are sequentially combined with thenon-aqueous solvent, in an order-independent manner, to form thephospholipid solution.

In some embodiments of the various aspects provided herein, the DPPC,DPPA and MPEG5000-DPPE stocks are combined with each other to form aphospholipid mixture and the phospholipid mixture is then combined withthe non-aqueous solvent to form the phospholipid solution. Thephospholipid mixture may be heterogeneous or homogeneous.

In some embodiments of the various aspects provided herein, the DPPC,DPPA and MPEG5000-DPPE stocks are combined with each other to form aphospholipid blend, and the phospholipid blend is combined with thenon-aqueous solvent to form the phospholipid solution. In someembodiments of the various aspects provided herein, the phospholipidblend is formed using an organic solvent dissolution-precipitationprocess comprising dissolving the DPPC, DPPA and MPEG5000-DPPE stocksinto a mixture of methanol and toluene, optionally concentrating thephospholipid/methanol/toluene mixture, and then contacting theconcentrated phospholipid/methanol/toluene mixture with methyl t-butylether (MTBE) to precipitate the phospholipids to form the phospholipidblend. In some embodiments of the various aspects provided herein, thephospholipid blend is formed by dissolving DPPC, DPPA and IMPEG5000-DPPEstocks into a blend solvent system, other than a methanol/toluenesolvent system, optionally concentrating the phospholipid/solventmixture, and then contacting the concentrated phospholipid solventmixture with methyl t-butyl ether (MTBE) to precipitate thephospholipids to form the phospholipid blend. In some embodiments of thevarious aspects provided herein, the phospholipid blend is formed bydissolving DPPC, DPPA and MPEG5000-DPPE stocks into a blend solventsystem, such as but not limited to a methanol/toluene solvent system,and then lyophilizing or otherwise drying the mixture to remove thesolvent, leaving behind the phospholipid blend.

In some embodiments of the various aspects provided herein, the methodFurther comprises placing the phospholipid suspension in a vial andintroducing a perfluorocarbon gas into the headspace of the vial.

In some embodiments of the various aspects provided herein, the methodfurther comprises activating the phospholipid suspension with theperfluorocarbon gas to form an ultrasound contrast agent comprisingphospholipid-encapsulated gas microspheres.

In some embodiments of the various aspects provided herein, the methodfurther comprises administering the ultrasound contrast agent to asubject and obtaining one or more contrast-enhanced ultrasound images ofthe subject.

In some embodiments of the various aspects provided herein, the methodfurther comprises measuring calcium concentration of the DPPA stockand/or DPPC stock and/or phospholipid mixture and/or phospholipid blend.

These and other aspects and embodiments of this disclosure will bedescribed in greater detail herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph of four phospholipid solutions having differingdegrees of precipitation. The Figure illustrates the appearance scaledefinition of), +, ++, and +++ as used in the Examples.

FIG. 2 is a photograph showing the appearance of solution uponsuccessive additions of DPPC, MPEG5000-DPPE, DPPA, and calcium acetate.

FIG. 3 is a photograph showing the appearance of the solutions for Study4 as described in the Examples.

FIG. 4 is a photograph showing the appearance of solutions prepared withdifferent proportions of MPEG5000-DPPE and MPEC35000-DPPE containinghigh calcium (Ca+2) levels.

FIG. 5 is a graph showing filtration flow rate for phospholipid aqueousformulation made with individual phospholipids cor phospholipid blendcontaining either high or low calcium. Studies 19 and 23 were made withlow calcium components whereas studies 20 and 24 contain high calciumlevels (see Table 6 for details). Each graphed point is an average ofthree consecutive filtration rate measurements with corresponding timewithin each study.

FIG. 6 is a graph showing filtration flow rate for phospholipidnon-aqueous formulation made with individual phospholipids orphospholipid blend containing either high or low calcium. Studies 33 and35 were made with low calcium components whereas studies 34 and 36contain high calcium levels (see Table 9 for details). Each graphedpoint is an average of three consecutive filtration rate measurementswith corresponding time within each study.

DETAILED DESCRIPTION

Provided herein are improved methods for preparing phospholipid-basedultrasound contrast agents (UCA). These improvements are based in parton the surprising discovery that certain phospholipid-basedformulations, intended for use in preparing ultrasound contrast agents,are susceptible to the presence and amount of certain divalent metalcations. Specifically, it was unexpectedly found that divalent metalcations, such as calcium, at certain concentrations, when introducedinto a phospholipid-based formulation used to generate the leadingultrasound contrast agent, DEFINITY®, caused phospholipid andpotentially other components of the formulation to precipitate out ofsolution, thereby rendering the formulation unusable. Such formulationsare typically made in large scale batches and thus the inadvertentaddition of calcium, for example, would render an entire batch unusable.This can lead to reduced manufacturing capability.

It has also been found, surprisingly, that certain phospholipids aremore susceptible to precipitation induced by the presence of divalentmetal cations such as calcium. Specifically, DPPA in non-aqueous solventsuch as propylene glycol is more likely to precipitate in the presenceof certain concentrations of divalent metal cations such as calcium.This same sensitivity was not observed, or not observed to the samedegree, with other phospholipids such as DPPC and DPPE. Thisdifferential precipitation profile can easily result in a phospholipidformulation, and ultimately a UCA, having a different phospholipidcomposition than planned or desired. Thus, not only can the presence ofdivalent metal cations reduce total yield of a UCA (e.g., due to thenon-filterability of a precipitate-containing phospholipid formulationsuch as the phospholipid suspensions described herein), it can alsointerfere with the phospholipid distribution of the UCA. This isproblematic because it may result in UCA formulations of wholly unknownphospholipid content. As is well known in the pharmaceutical arts, thecomposition of such UCA formulations must remain constant and robustlyreproducible, and hatch-to-hatch variability must be avoided orminimized to the greatest extent possible.

This disclosure therefore provides improved methods for preparingphospholipid formulations such as phospholipid solutions andphospholipid suspensions, as described herein. These methods improve theyields of such formulations by reducing the likelihood of phospholipidprecipitation. They also produce, in a more robust and reproduciblemanner, phospholipid formulations having their intended phospholipidprofiles and distributions. These methods take advantage of the noveland surprising findings described herein and provide phospholipidformulations of the desired phospholipid content and proportion withoutresorting to detecting precipitate.

Phospholipid Formulations, Generally

Provided herein are methods for preparing improved phospholipidsolutions, phospholipid suspensions and ultimately UCA formulations. Aswill be described in greater detail, in some instances, the UCAformulations may be formed from non-aqueous phospholipid solutions thatare combined with a gas such as perflutren. In other instances, the UCAformulation may be formed from combining the non-aqueous phospholipidsolution with an aqueous solvent to form a phospholipid suspension thatis combined with a gas such as perflutren. These and otherphospholipid-containing compositions are collectively referred to hereinas phospholipid formulations. Each of the specific formulations will bedescribed in greater detail below. The phospholipid formulations of thisdisclosure may comprise the three phospholipids that are used in themanufacture of the FDA-approved DEFINITY® microspheres. These threephospholipids are

-   -   (1) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (referred        to herein as DPPC),    -   (2) 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (referred to        herein as DPPA), and    -   (3) N-(methoxy polyethylene glycol 5000        carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine        (referred to herein as MPEG5000-DPPE).

The phospholipid formulations of this disclosure may comprise DPPC andMPEG-5000-DPPE.

In some instances, modified forms of one or more of these phospholipidsmay be used. For example,1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) may beconjugated to polyethylene glycol (PEG). The PEG conjugated to DPPE, orto another phospholipid, may have a molecular weight (MW, or length)selected from 1000-10,000, in some, non-limiting instances. Moretypically, the PEG may have a MW of about 5000, in which case it isreferred to as PEG5000, and when conjugated to DPPE is referred to asPEG5000-DPPE. The PEG is typically conjugated to a phospholipid such asDPPE at the phospholipid head group rather than at the aliphatic chainend. The PEG may have a hydroxy or a methoxy terminus, and may bereferred to as HO-PEG5000 or as MPEG5000, respectively.

When conjugated to a DPPE, as an example, the conjugate may be referredto as HO-PEG5000-DPPE or as MPEG5000-DPPE. The full chemical name of thelatter conjugate is N-(methoxy polyethylene glycol 5000carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, monosodium salt (referred to herein as MPEG5000-DPPE).

DPPA, DPPC and MPEG5000-DPPE may be used in molar percentages of about77-90 mole % DPPC, about 5-15 mole % DPPA, and about 5-1.5 mole % DPPE,including MPEG5000-DPPE. Preferred ratios of each phospholipid includeweight % ratios of 6.0 to 53.5 to 40.5 (DPPA DPPC MPEG5000-DPPE) or amole % ratio of 10 to 82 to 8 (10:82:8) (DPPA:DPPC:MPEG5000-DPPE).

The remainder of this disclosure will refer specifically to DPPA, DPPCand MPEG5000-DPPE for convenience and brevity, but it is to beunderstood that the teachings provided herein are intended to encompassmethods that utilize and/or compositions that comprise these or otherphospholipids singly or in combination such as but not limited to acombination of DPPC, and MPEG5000-DPPE.

Various methods provided herein involve measuring the divalent metalconcentration of the components used to make the phospholipidformulations described herein. Of particular importance are thecomponents used to make the phospholipid solutions, particularly sinceprecipitation appears to be a phenomenon first observed at thephospholipid solution step rather than at the phospholipid suspensionstep. Methods that may be used to measure divalent metal cationconcentration, such as calcium and magnesium concentration, aredescribed in greater detail herein including in the Examples. Somemethods may involve measuring the divalent metal cation concentration ofonly one component, such as for example MPEG5000-DPPE. Other methods mayinvolve measuring the divalent metal cation concentration of two or moreof the components such as for example two or three of the phospholipids.In some embodiments, the components may be combined together before themeasurement is made. Still other methods involve measuring the divalentmetal cation concentration of all the components, including thenon-aqueous solvent, used to make the phospholipid formulation such asthe phospholipid solution. Such measurement may be made before or afterthe components are combined. For example, measurement may be made ofindividual components used to make a phospholipid solution or it may bemade of the phospholipid solution itself.

Various other methods provided herein involve selecting components usedto make the phospholipid formulations such as the phospholipidsolutions, based on their divalent metal cation concentration. Morespecifically, the methods involve selecting one or more components thathave been characterized or identified as having no or low divalent metalcation concentration, including no or low calcium concentration or no orlow magnesium concentration. Some methods may involve selecting onecomponent, such as MPEG5000-DPPE, characterized or identified as havingno or low divalent metal cation concentration, including no or lowcalcium concentration or no or low magnesium concentration. Some methodsmay involve selecting two or more or all components based on theircombined divalent metal cation concentration. Thus, it is contemplatedthat DPPA, DPPC and MPEG5000-DPPE, as well as other components such asbut not limited to non-aqueous solvent and/or its individual components,may be individually characterized as having no or low divalent metalcation concentration but that when used together their combined divalentmetal cation concentration will no longer satisfy the requirement of noor low divalent metal cation concentration and will cause precipitation.Thus, in these and other instances, two, three or all of the componentssuch as two or three of the phospholipids may be selected such thattheir combined divalent metal cation concentration is characterized asno or low divalent metal cation concentration.

Phospholipid Solution

As used herein, a phospholipid solution refers to a compositioncomprising one or more phospholipids in a non-aqueous solvent. Thephospholipid solution may minimally comprise DPPA, DPPC, andMPEG5000-DPPE in a non-aqueous solvent. The phospholipid solution mayminimally comprise DPPC and MPEG5000-DPPE in a non-aqueous solvent.

A non-aqueous solvent, as used herein, is a solvent that causesphospholipids to dissolve thereby forming solution (i.e., a phospholipidsolution). Preferably, the non-aqueous solvent present in thephospholipid. solution is pharmaceutically acceptable, particularlysince it is carried through to the final VCR formulation that isadministered to a subject including human subject. In certainembodiments, the non-aqueous solvent used to make the phospholipidsolution is not or does not comprise methanol or toluene or methylt-butyl ether (MTBE).

The non-aqueous solvent of the phospholipid solution may be a singlesolvent or it may be combination of solvents. Non-aqueous solventsinclude but are not limited to propylene glycol (which may be referredto herein as PG) and glycerol (which may be referred to herein as G).Both are provided as liquid stocks. In some instances, the non-aqueoussolvent of the phospholipid solution may be PG alone or it may be amixture of PG and G (which may be referred to as PG/G). A non-aqueoussolvent that comprises at least PG may be referred to herein as aPG-comprising non-aqueous solvent. The PG/G mixtures include ratiosranging from 5:1 to 1:5 (weight by weight). In some embodiments, a PG:Gw/w ratio of 1:1 is used (and is referred to herein as a 1:1 mixture).

The phospholipid solution may further comprise one or more buffers. Suchbuffers are those capable of buffering a non-aqueous solvent such asthose recited above. Examples include, without limitation, an acetatebuffer (e.g., a combination of sodium acetate and acetic acid), abenzoate buffer (e.g., a combination of sodium benzoate and benzoicacid), and a salicylate buffer (e.g., a combination of sodium salicylateand salicylic acid). Other buffers that may be used include adiethanolamine buffer, a triethanolamine buffer, a borate buffer, acarbonate buffer, a glutamate buffer, a succinate buffer, a malatebuffer, a tartrate buffer, a glutarate buffer, an aconite buffer, acitrate buffer, a lactate buffer, a glycerate buffer, a gluconatebuffer, and a tris buffer. In some embodiments, an acetate buffer isused. The buffer used in the non-aqueous solvent may be a non-phosphatebuffer intending that it is not a phosphate buffer.

The buffer concentration will vary depending on the type of buffer used,as will be understood and within the skill of the ordinary artisan todetermine. The buffer concentration in the non-aqueous solvent may rangefrom about 1 mM to about 100 mM, including about 1 mM to about 50 mM, orabout 1 mM to about 20 mM, or about 1 mM to about 10 mM, or about 1 mMto about 5 mM, including about 5 mM.

Accordingly, the phospholipid solution may comprise one or morephospholipids such as DPPA, DPPC and MPEG5000-DPPE, a non-aqueoussolvent that is or that comprises PG, and optionally a buffer such asacetate buffer.

The phospholipid solution may be made in a number of ways, several ofwhich are described in greater detail below. In general, the non-aqueoussolvent may be warmed prior to contact with the phospholipids, and ifused the buffer may first be present in the solvent prior to contactwith the phospholipids. The solvent and then solution may be stirred tofacilitate dissolution of the phospholipids.

Significantly, it has been found that phospholipid precipitationassociated with divalent metal cations occurs in the non-aqueous solventand thus in the process of making the phospholipid solution. Thus, asdescribed herein various methods include steps of measuring divalentmetal cation concentration of the various components used to make thephospholipid solution, including the phospholipids whether individuallyor collectively, the non-aqueous solvent such as the PG and G, thebuffer such as the acetate buffer, if used, and the like.

In some embodiments, the divalent metal cation concentration of thephospholipid suspension may be measured, instead of or in addition tomeasuring the divalent metal cation concentration of the phospholipidsolution.

A visual observation of the phospholipid solution may be made to detectprecipitate, although this is not required. FIG. 1 is a photographshowing various phospholipid solutions having differing degrees ofprecipitate.

In some embodiments, the phospholipid solution is then used to preparethe phospholipid suspension described in greater detail below.

In some embodiments, the phospholipid solution is directly contactedwith gas such as a perfluorocarbon gas to make phospholipid encapsulatedgas microspheres, without first contacting the phospholipid solutionwith an aqueous solvent. That is, in some instances, thephospholipid-encapsulated gas microspheres are made through contact andvigorous shaking (referred to as activation) of the (non-aqueous)phospholipid solution and the gas. Such microspheres may then becontacted with an aqueous solvent to form a UCA.

Phospholipid Suspension

As used herein, a phospholipid suspension refers to an aqueousphospholipid formulation comprising phospholipid solution and an aqueoussolvent. The phospholipid suspension may comprise one or morephospholipids such as DPA, DPPC and MPEG5000-DPPE. A phospholipidsuspension will minimally comprise one or more phospholipids such as oneor more phospholipids, a non-aqueous solvent such as PG, and an aqueoussolvent.

An aqueous solvent, as used herein, is or comprises water as its majorcomponent (by weight). An aqueous solvent may further comprise one ormore salts, and thus may be referred to as an aqueous saline solvent, itmay additionally or alternatively comprise a buffer, and thus may bereferred to as an aqueous buffered saline solvent or an aqueous bufferedsolvent. Preferably, the aqueous solvent, regardless of whether itincludes salt(s) or buffer(s) is pharmaceutically acceptable, since likethe phospholipid solution it is carried through to the final UCAformulation that is administered to a subject including a human subject.

Salts that may be included in the aqueous solvent include but are notlimited to sodium chloride.

Buffers that may be included in the aqueous solvent include but are notlimited to phosphate buffer, acetate buffer, benzoate buffer, salicylatebuffer, diethanolamine buffer, triethanolamine buffer, borate buffer,carbonate buffer, glutamate buffer, succinate buffer, malate buffer,tartrate buffer, glutarate buffer, aconite buffer, citrate buffer,lactate buffer, glycerate buffer, gluconate buffer, and a tris(tris(hydroxymethyl)methylamine) buffer. Typically, either thenon-aqueous solvent or the aqueous solvent comprises a buffer, but notboth. The buffer concentration will vary depending on the type of bufferused, as will be understood and within the skill of the ordinary artisanto determine. The buffer concentration in the aqueous solvent may rangefrom about 1 mM to about 100 mM, including about 1 mM to about 50 mM, orabout 10 mM to about 30 mM, or about 20 mM to about 30 mM or about 20 mMto about 25 mM, including about 25 mM.

Accordingly, the phospholipid suspension may comprise one or morephospholipids such as DPPA, DPPC and MPEG5000-DPPE, a non-aqueoussolvent that is or that comprises PG, an aqueous solvent that maycomprise one or more salts such as sodium chloride, and optionally abuffer such as acetate buffer or a phosphate buffer. Phospholipidsuspensions may be physically characterized as phospholipids suspended,rather than dissolved, in an aqueous solvent.

The phospholipid suspension is generally made by contacting thephospholipid solution, which is non-aqueous, with the aqueous solvent.The aqueous solvent may already comprise any salts and/or any buffers oralternatively those may be added after contact with the phospholipidsolution. The aqueous solvent may be stirred in order to ensure mixingof the phospholipid solution with the aqueous solvent. The aqueoussolvent may also be warmed prior to contacting with phospholipidsolution which in some instances may also be warmed.

Surprisingly, the divalent metal cation concentration of the aqueoussolvent is not as important as that of the non-aqueous phospholipidsolution (and its combined components). For example, it has been foundunexpectedly that once a precipitate-free phospholipid solution isprepared, it can be combined with an aqueous solvent that has a highdivalent metal cation concentration without inducing any discernablephospholipid precipitate. Thus, it has been found surprisingly that thephospholipid sensitivity to high divalent metal cation content existsonly in the phospholipid solution or in the presence of the non-aqueoussolvent, but not beyond that point. Similarly, it has been found thatonce the precipitate is formed in the phospholipid solution, contactwith the aqueous solvent, even if warmed, does not lead to itsdissolution. This differential sensitivity of phospholipids, and inparticular, DPPA to high divalent metal cation levels, such as calciumlevels, was not heretofore appreciated and was considered a surprisingfinding.

While the divalent metal cation concentration of the aqueous solventdoes not appear to induce precipitation of one or more phospholipids, itdoes surprisingly induce precipitation of other components, includingmost notably phosphate such as may be present if a phosphate buffer isused in the aqueous solvent. Thus, in some instances, the methodsprovided herein may further include measuring divalent metal cationconcentration of components used to make the aqueous phospholipidsuspensions that comprise phosphate. Alternatively, the methods mayinclude selecting individual components or combined components that arecharacterized, individually or in combination, as having no or lowdivalent metal cation concentration.

The phospholipid suspension may then be used to prepare thephospholipid-encapsulated gas microspheres.

Phospholipid-encapsulated Gas Microspheres, and UCA for FormulationsComprising Them

As will be apparent, the phospholipid-based ultrasound contrast agentsof this disclosure are phospholipid-encapsulated gas microspheres. Thesemicrospheres may be made in a number of ways. For example, aphospholipid solution may be contacted with an aqueous solvent to form aphospholipid suspension, and the phospholipid suspension may becontacted with a gas such as a perfluorocarbon gas to form thephospholipid-encapsulated gas microspheres. As another example, thenon-aqueous phospholipid solution may be contacted with a gas such as aperfluorocarbon gas to form the phospholipid-encapsulated gasmicrospheres. In either instance, the phospholipid formulation, be it anon-aqueous phospholipid solution or an aqueous phospholipid suspension,is combined with the gas in a manner sufficient to create thephospholipid-encapsulated gas microspheres. This usually involvesvigorous shaking or other agitation. Sufficient shaking or agitation istypically achieved using a device, such as a VIALMIX®, and is nottypically achieved manually.

The phospholipid solution or the phospholipid suspension are provided ina container, such as a vial, having a gas headspace. A perfluorocarbongas, such as perflutren, is introduced into the headspace of suchcontainers, usually through a process of gas exchange. It is this vialthat is then vigorously shaken in order to form thephospholipid-encapsulated gas microspheres. This process, known asactivation, is carried out by the end user or medical personnel justprior to administration into a subject.

The microspheres comprise gas, such as a perfluorocarbon gas includingbut not limited to perflutren gas, in their internal cavity. Thephospholipid shell that encapsulates the gas may be arranged as aunilayer or a bilayer, including unilameilar or multilamellar bilayers.The microspheres may have a mean diameter of less than 10 microns, orless than 6 microns, or less than 3 microns, or more preferably lessthan 2 microns. These mean diameters intend that, when a population ofmicrospheres is analyzed, the mean diameter of the population is lessthan 10 microns, or less than 6 microns, or less than 3 microns, or morepreferably less than 2 microns. The microspheres may have a meandiameter in the range of 0.5 to 3 microns, or 1 to 2 microns, or 1.4 to1.8 microns, or 1.4 to 1.6 microns. The mean diameter may be about 1.4microns.

The process of generating phospholipid-encapsulated gas microspheres isknown as activation. Formulations that comprise a sufficientconcentration of phospholipid-encapsulated gas microspheres may bereferred to herein as activated formulations.

It will be appreciated that the concentration of the gas microspheresthat is “sufficient” will depend on whether the gas microspheres aremade using the phospholipid solution (without intervening use of anaqueous solvent) or are made using the phospholipid suspension.Typically, the UCA formulation being administered to a subject willcomprise on the order of about at least 1×10⁷ microspheres per ml ofadministered formulation, or at least 5×10⁷ microspheres per ml, or atleast 7.5×10⁷ microspheres per ml, or at least 1×10⁸ microspheres perml, or at least 1×10⁹ microspheres per ml, or about 5×10⁹ microspheresper ml. The range of microsphere concentration may be, in someinstances, 1×10⁷ to 1×10¹⁰ microspheres per ml of administeredformulation, and more typically 5×10⁷ to 5×10⁹ microspheres per ml.

Depending on how they are made, the gas microspheres may be present in anon-aqueous solvent or in an aqueous solvent. Regardless, prior toadministration to a subject, they are typically diluted in an aqueoussolution that may be a saline solution, or a buffered aqueous solution,or a buffered saline solution.

The UCA formulation to be administered, typically intravenously, to asubject including a human subject may have a pH in the range of 4-8 orin a range of 4.5-7.5. In some instances, the pH may be in a range ofabout 6 to about 7.5, or in a range of 6.2 to about 6.8. In still otherinstances, the pH may be about 6.5 (e.g. 6.5+/−0.5 or +/−0.3). In someinstances, the pH may be in a range of 5 to 6.5 or in a range of 5.2 to6.3 or in a range of 5.5 to 6.1 or in a range of 5.6 to 6 or in a rangeof 5.65 to 5.95. In still another instance, the pH may be in a range ofabout 5.7 to about 5.9 (e.g., +/−0.1 or +/−0.2 or +/−0.3 either or bothends of the range). In another instance, the pH may be about 5.8 (e.g.,5.8+/−0.15 or 5.8+/−0.1).

The gas is preferably substantially insoluble in the phospholipidformulations provided herein, including the phospholipid solution andthe phospholipid suspension. The gas may be a non-soluble fluorinatedgas such as sulfur hexafluoride or a perfluorocarbon gas. Examples ofperfluorocarbon gases include perfuoropropane, perfluoromethane,periluoroethane, perfluorohutane, perfluoropentane, perfluorohexane.Examples of gases that may be used are described in U.S. Pat. No.5,656,211 and are incorporated by reference herein. In an importantembodiment, the gas is perfluoropropane.

Divalent Metal Cations, and Methods of Measuring Same

Divalent metal cations are divalent metal ions with a valence of 2.These include: barium(2+), beryllium(2+), cadmium(2+), calcium(2+),chromium(2+), cobalt(2+), copper(2+), europium(2+), gadolinium(2+),germanium(2+), iron(2+), lanthanum(2+), lead(2+), magnesium(2+),manganese(2+), mercury(2+), nickle(2+), osmium(2+), platinum(2+),ruthenium(2+), strontium(2+), tin(2+), uranium(2+), vanadium(2+),yttrium(2+), and zinc(2+).

In some embodiments, the divalent metal cations of interest are calcium,magnesium and manganese. In some embodiments, the divalent metal cationsof interest are calcium and magnesium and therefore only calcium andmagnesium are measured or components are selected based only on theircalcium and magnesium content. In some embodiments, the divalent metalcation of interest is calcium, and therefore only calcium is measured orcomponents are selected based on their calcium concentration.

Effect of Divalent Metal Cations

As described herein, divalent metal cations may be present in one ormore of the components used to make the UCA formulations. Their presencemay not be appreciated until such components are combined with thenon-aqueous solvent to form the phospholipid solution, at which pointphospholipid precipitation may be induced For example, or until suchcomponents are combined with the aqueous solvent to form thephospholipid suspension, at which point phosphate precipitation may beinduced for example. Surprisingly, it was discovered in accordance withthis disclosure that the MPEG5000-DPPE phospholipid stock containedcalcium and magnesium at sufficiently high concentrations to causeprecipitation of at least the DPPA phospholipid once combined. Thus, thedivalent metal cations may have different effects on differentphospholipids, and it may not be readily apparent to the user whether aphospholipid (or other component) contains such cations atconcentrations sufficient to induce precipitation.

The inventors discovered in the process of preparing various UCAformulations that a non-aqueous solvent became cloudy when combined witha phospholipid blend comprising DPPA, DPPC and MPEG5000-DPPEphospholipids. It was further determined that the cloudy appearance waslikely due to the precipitation of the DPPA phospholipid. Unbeknownst tothe inventors, however, was the fact that the MPEG5000-DPPE containedhigh concentrations of calcium and magnesium ions and that those calciumand magnesium concentrations were likely the cause of the DPPAprecipitation. Interestingly, such cations did not appear to affect theability of MPEG5000-DPPE to remain in solution and thus a user would notappreciate that fact until such phospholipid was combined with theothers in the non-aqueous mixture. Further studies, described in greaterdetail herein, found that precipitation occurred when a MPEG5000-DPPEstock later characterized as having a high calcium concentration wascombined with DPPA, regardless of the order of addition or the presenceof other components such as other phospholipids such as DPPC and DPPA.The sensitivity of DPPA to precipitate in a non-aqueous solventcomprising PG in the presence of sufficiently high divalent metal cationconcentration such as calcium and magnesium concentration yet not in anaqueous solvent having similarly high concentrations of either or bothcalcium and magnesium was even more surprising. In other words, theconcentrations of calcium that caused DPPA precipitation in non-aqueoussolvent comprising PG did not cause DPPA precipitation in the aqueoussolvent, and this too was surprising.

No or Low Divalent Metal Cation Concentration

As used herein, components are selected that are characterized oridentified as having no or low divalent metal cation concentration,which includes no or low calcium concentration. Such divalent metalcation concentration is expressed as a weight by weight measure (i.e.,weight of the divalent metal cation per unit weight of the underlyingmatrix or solvent in which the component of interest is present). Amicrogram per grain concentration may be alternatively referred to asparts per million or ppm.

A no or low calcium concentration of such component will further dependupon how much of that component is used or in other words how much suchcomponent is diluted to form the phospholipid solution or thephospholipid suspension.

In the simplest case, only one component is of interest, and only itscalcium concentration is measured or only that component is selectedbased on its calcium concentration. Based on this disclosure, one ofordinary skill in the art will understand and be able to determine howmuch calcium will be tolerated in that component in order to avoidprecipitation in the phospholipid solution or the phospholipidsuspension.

As an example, calcium concentration in a phospholipid stock (which istypically provided as a solid such as a powder) is expressed in weightof calcium per gram of phospholipid. An example is a calcium weight pergram of MPEG5000-DPPE or calcium weight per gram DPPC. When twocomponents such as two phospholipids are combined, the measure may beweight of calcium per gram of MP; G5000-DPPE and. DPPC combined.

No divalent metal cation, such as no calcium, refers to a concentrationof such cation that is undetectable using the methods known in the artand/or provided herein.

No or low divalent metal cation concentration in a phospholipid stockwill depend on the particular component.

No or low divalent metal cation concentration in an MPEG5000-DPPEphospholipid stock is less than 510 micrograms/gram (i.e., micrograms ofdivalent metal cation per gram of WIPEG-5000-DPPE) (also referred to asless than 510 ppm), including less than 345 ppm, less than 230 ppm, lessthan 115 ppm, less than 57.5 ppm, and less than 11.5.

No or low divalent metal cation concentration in a DPPC phospholipidstock is less than 390 micrograms/gram (i.e., micrograms of divalentmetal cation per gram of DPPC) (also referred to as less than 390 ppm),including less than 270 ppm, less than 180 ppm, less than 90 ppm, lessthan 45 ppm, and less than 9 ppm.

No or low divalent metal cation concentration in a DPPA phospholipidstock is less than 3440 micrograms/gram (i.e., micrograms of divalentmetal cation per gram of DPPA) (also referred to as less than 3440 ppm),including less than 2340 ppm, less than 1560 ppm, less than 780 ppm,less than 390 ppm, and less than 78 ppm.

No or low divalent metal cation concentration in a phospholipid blend isless than 210 micrograms/gram (i.e., micrograms of divalent metal cationper gram of phospholipid blend or the combined weight of MPEG5000-DPPEand DPPC and DPPA) (also referred to as less than 210 ppm), includingless than 150 ppm, less than 100 ppm, less than 50 ppm, less than 25ppm, and less than 5 ppm.

No or low divalent metal cation concentration in propylene glycol isless than 3.1 micrograms/gram (i.e., micrograms of divalent metal cationper gram of propylene glycol) (also referred to as less than 3.1 ppm),including less than 2.1 ppm, less than 1.4 ppm. less than 0.7 ppm, lessthan 0.35 ppm, and less than 0.07 ppm.

No or low divalent metal cation concentration in a 1:1 (weight toweight) propylene glycol and glycerol mixture is less than 10.4micrograms/gram (i.e., micrograms of divalent. metal cation per gram ofpropylene glycol and glycerol combined) (also referred to as less than10.4 ppm), including less than 7.8 ppm, less than 5.2 ppm, less than 2.6ppm, less than 1.3 ppm, and less than 0.26 ppm.

No or low divalent metal cation concentration in glycerol is less than20.4 micrograms/gram (i.e., micrograms of divalent metal cation per gramof glycerol) (also referred to as less than 20.4 ppm), including lessthan 15.3 ppm, less than 10.2 ppm, less than 5.10 ppm, less than 2.6ppm, less than 0.51 ppm.

No or low divalent metal cation concentration in a phospholipid solutionthat comprises only propylene glycol as the non-aqueous solvent is lessthan 3.1 micrograms/gram (i.e., micrograms of divalent metal cation pergram of all components of the phospholipid solution) (also referred toas less than 3.1 ppm), including less than 2.1 ppm, less than 1.4 ppm.less than 0.7 ppm, less than 0.35 ppm, and less than 0.07 ppm As will beappreciated based on the composition of the phospholipid solution, themajor component by weight is the non-aqueous solvent, in this particularcase, propylene glycol.

It is to be understood that the same concentration limits apply tocalcium concentration and magnesium concentration, as well as combinedcalcium and magnesium concentrations.

This disclosure contemplates measurement of divalent metal cationconcentration in one or more components of the phospholipid solution,including for example one, two or all three of the phospholipid stocks,optionally together with measurement of divalent metal cationconcentration of the non-aqueous solvent such as propylene glycol and/orglycerol, depending on the nature of the phospholipid solution. If anyof these components contain a divalent metal cation concentration inexcess of those levels recited above, then it is expected that once aphospholipid solution is made with such component, such phospholipidsolution will be prone to precipitation.

Some embodiments contemplate that a single component, will be analyzedfor its divalent metal cation concentration and that if suchconcentration is a “no or low divalent metal cation concentration” thenthe component may be combined with the remaining components even if suchcomponents were not analyzed for their divalent, metal cationconcentration. Components of a phospholipid solution include thephospholipid stocks, whether provided individually or as a blend,non-aqueous solvents such as propylene glycol and glycerol, andoptionally buffers.

Some embodiments contemplate that more than one but less than allcomponents of the phospholipid solution will be measured for theirdivalent metal cation concentration. In some instances, if one of thecomponents have a divalent metal cation concentration that is in excessof the “no or less divalent metal cation concentration” limit listedabove, then it may not be used to prepare the phospholipid solution (orphospholipid blend). In some instances, no one component may becharacterized or identified as having a divalent metal cationconcentration that is more than the “no or low divalent metal cationconcentration”. However, when such components are used in combination,the combined divalent metal cation concentration may be determined basedon the amount each contributes to the phospholipid solution. It iscontemplated that the combined divalent metal cation concentration mayor may not exceed the “no or low divalent metal cation concentration” asdefined above for the phospholipid solution, As discussed throughout,the various levels set forth herein while referring to divalent metalcations, apply equally to calcium. The Examples demonstrate that thelowest calcium concentration at which precipitation of phospholipid isapparent is about 0.7 microgram calcium per gram of non-aqueous solvent(see Examples 1 and 2), otherwise referred to as about 0.7 ppm. Thus, ano or low divalent metal cation concentration such as a no or lowcalcium concentration in a non-aqueous phospholipid solution is lessthan 0.7 ppm, less than 0.35 ppm, or less than 0.07 ppm. It is to beunderstood that a divalent metal cation concentration in thephospholipid solution of less than 0.7 ppm will also be regarded as a noor low divalent metal ion concentration.

Divalent metal cation concentration in an aqueous phospholipidsuspension may be provided as a weight of divalent metal cation per gramof aqueous solvent. Typically, the phospholipid suspension is formed bydiluting the non-aqueous phospholipid solution about 20-fold into theaqueous solvent. Thus, a no or low divalent metal ion concentration in aphospholipid suspension is less than 0.035 micrograms per gram ofphospholipid suspension. Similarly, the calcium concentration in aphospholipid suspension originating from the phospholipid solution isless than 0.035 micrograms per grain of phospholipid suspension.

The concentration at which the divalent metal cations causephospholipids to precipitate may be temperature dependent. At highertemperatures, higher concentrations of cations may be tolerated beforeprecipitation is observed. At lower temperatures, lower concentrationsof cations may cause the precipitation to occur.

As an example, at temperatures of about 55° C. (e.g., 50-60° C.), no orow divalent metal cation concentration is a divalent metal cationconcentration of less than 0.7 micrograms calcium per gram ofphospholipid solution or of non-aqueous solvent. This level may beslightly lower if the phospholipid solution is prepared at a lowertemperature. Alternatively, this level may be slightly higher if thephospholipid solution is prepared at a higher temperature.

Calcium Sources

As demonstrated in the Examples, the phospholipid solution isunexpectedly and uniquely sensitive to particular levels of calcium.This unique sensitivity was not heretofore recognized. Given the effectof calcium on the preparation of the phospholipid solution, and thusultimately on the UCA, it is important to measure and thus control thecalcium concentration of the phospholipid solution. Calcium may bepresent in each of the components of the phospholipid solution,including the phospholipid stocks and the non-aqueous solvents, asdescribed below.

Calcium and magnesium are divalent alkaline earth metals in group 2 ofthe periodic table. Calcium is the fifth-most-abundant element by massin the Earth's crust and the cation Ca2+ is also the fifth-most-abundantdissolved ion in seawater. It is found at various levels in tap waterdepending on the on the “hardness”. The total water hardness is the sumof the molar concentrations of Ca2+ and Mg2+, ranging from soft at 0-60ppm to very hard≥181.

Calcium and magnesium are also found in varying concentration in thecrude glycerol extract from biodiesels. Level of calcium and magnesiumwere reported to range from 12. to 163 ppm and 4 to 127 ppm respectivelydepending on the seed oil for biodiesel production (J. C. Thompson 2006Applied Engineering in Agriculture Vol. 22(2): 261-265). A major supplyof glycerol comes from this biodiesel byproduct. The crude glycerolextract can be purified by treatment with activated carbon to removeorganic impurities, alkali to remove unreacted glycerol esters, and ionexchange to remove salts. High purity glycerol (>99.5%) is obtained bymulti-step distillation; vacuum is helpful due to the high boiling pointof glycerol (290° C.).

Industrially, propylene glycol is produced from propylene oxide.Different manufacturers use either non-catalytic high-temperatureprocess at 200° C. (392° R.) to 220° C. (428° F.), or a catalyticmethod, which proceeds at 150° C. (302° F.) to 180° C. (356° F.) in thepresence of ion exchange resin or a small amount of sulfuric acid oralkali. Final products contain 20% propylene glycol, 1.5% of dipropyleneglycol and small amounts of other polypropylene glycols. Furtherpurification produces finished industrial grade or USP/JP/EP/BP gradepropylene glycol that is typically 99.5% or greater, Propylene glycolcan also be converted from glycerol, a bio diesel byproduct.

The calcium and magnesium contents of Pharmacopeia grade propyleneglycol and glycerol are not quantified as a certificate of analysisrequirement of US Pharmacopeia, European Pharmacopeia, BritishPharmacopeia or Japanese Pharmacopeia.

Phospholipid DPPA contains a phosphate which can be ionized atappropriate pH. The pKa for the two hydroxyl groups of the phosphate are6.2 and 1.8 (Tatulian Ionization and Binding, 511-552 PhospholipidHandbook, Ed. G Cove 1993). DPPA is commercially available as differentsalt forms. Usually, the Na salt is used but the Ca salt is alsoavailable,

DPPC is a zwitterion and therefore does not require a counter ion.

MPEG5000-DPPE is a modified DPPE which has a pKa of 1.9 and 9.3 for thehydroxyl of the phosphate and the amine of the ethanolamine (TatulianIonization and Binding, 511-552 is Phospholipid Handbook, Ed. G Ceve1993). MPEG500-DPPE is available as the Na salt form,

Methods of Measuring Divalent Metal Cation Concentration

Quantitation of divalent metal cations concentration can be performedusing one of several known techniques. These include atomic spectroscopymethods such as atomic absorption spectroscopy (AAS), flame photometryor flame atomic emission spectrometry (FAES), inductively coupledplasma-atomic emission spectrometry (ICP-AES), and other methods such asinductively coupled plasma-mass spectroscopy or complexometrictitration. The spectroscopic approaches utilize absorption or emissioncharacteristics of the metal ions of alkali metals (Group I) andalkaline earth metals (Group II) metals when dissociated due to thermalenergy provided by a flame source. ICP-MS is a type of mass spectrometrywhich is capable of detecting metals at very low concentrations. This isachieved by ionizing the sample with inductively coupled plasma and thenusing a mass spectrometer to separate and quantify those ions. Sonic ofthese methods are used in the Examples.

Complexometric titration is another method for detecting divalent metalcation concentration. This method uses EDTA (ethylenediaminetetraaceticacid) complexation with calcium and magnesium ions to compete with acolor indicator. This allows rapid colorimetric quantitation. EDTA formsa complex with calcium and magnesium ions. A blue dye called EriochromeBlack T (ErioT) is used as the indicator. This blue dye also forms acomplex with the calcium and magnesium ions, changing color from blue topink in the process. The dye-metal ion complex is less stable than theEDTA-metal ion complex. For the titration, the sample solutioncontaining the calcium and magnesium ions is reacted with an excess ofEDTA. The indicator is added and remains blue as all the Ca2+ and Mg2+ions present are complexed with the EDTA.

A back titration is carried out using a solution of magnesium chloride.This forms a complex with the excess EDTA molecules until the end-point,when all the excess EDTA has been complexed. The remaining magnesiumions of the magnesium chloride solution then start to complex with ErioTindicator, immediately changing its color from blue to pink

Methods of Synthesis

The disclosure provides methods for preparing phospholipid solutions andphospholipid suspensions intended for use with a perfluorocarbon gas toform a UCA formulation comprising phospholipid-encapsulated gasmicrospheres. In preferred embodiments, the phospholipids are DPPA, DPPCand DPPE such as MPEG5000-DPPE.

The phospholipid solution may be made in a number of ways, as describedbelow. These methods are characterized broadly as blend and non-blendmethods. The starting phospholipid stocks may be in solid (e.g., powder)form or in liquid form.

Blend Methods

Blend methods refer to methods in which the phospholipids are intimatelyblended with each other in order to render a solid phospholipid mixturethat is more uniform (and thus more homogenous) with respect to itsphospholipid content and phospholipid distribution and in some instanceshas higher purity, as compared to simple mixtures of phospholipids.

This method creates a homogenous dispersion of the three phospholipidsby dissolving or suspending them in an appropriate blend solvent system,and then separates the evenly distributed phospholipids from thesolvent. The separation of the blend solvent from the phospholipids caninvolve drying, lyophilization, distillation, and the like, or it caninclude precipitation using an additional blend solvent. Blend solventsfor neutral lipids are relatively non-polar solvents such as diethylether or chloroform. More polar blend solvents such as alcohol (e.g.,methanol and ethanol) are required for membrane-associated lipids whichare themselves more polar. Chloroform may also be used, particularly forlipids of intermediate polarity. When mixed with methanol, chloroformbecomes a general solvent. Dichloromethane (or methylene dichloride) isa similar extractant but less oxidizable. Hexane may be used for lipidsof low polarity. It can be used to extract neutral lipids fromwater/alcohol mixtures. Petroleum ether is a mixture of varioushydrocarbons with 5-8 carbon atoms and may be used in place of hexanesin some instances. Other blend solvents include without limitationcyclohexane and toluene.

As will be described in greater detail herein, certain blends are formedby contacting one or more desired phospholipids (e.g., DPPA, DPPC andMPEG5000-DPPE) in a blend solvent system to first dissolve suchphospholipids, optionally then concentrating such solution, then eitherremoving the blend solvent or precipitating the phospholipid blend fromsuch solvent. The blend solvent is not to be confused with thenon-aqueous solvent that is later used to dissolve the phospholipids,thereby forming a phospholipid solution. It should also be clear thatthis precipitation is a desired event and is not to be confused with theundesirable phospholipid precipitation that can occur during the laterstep of forming the phospholipid solution by the presence of calcium,another divalent cation, or a combination of divalent cations.

Some methods of preparing phospholipid solutions involve contacting aphospholipid blend with the non-aqueous solvent. There are various waysof making phospholipid blends, including but not limited to organicsolvent (or blend solvent, as used herein) dissolution-precipitationmethods and aqueous suspension-lyophilization methods.

The organic solvent dissolution-precipitation method is described indetail in U.S. Pat. No. 8,084,056 and in published InternationalApplication No. WO99/36104, the entire contents of both of which areincorporated herein by reference. One embodiment of this method involvesthe following steps:

(a) Contacting the desired phospholipids (e.g., DPPA, DPPC andMPEG5000-DPPE or DPPC and MPEG5000-DPPE) with a first blend solventsystem. This system is typically a combination. of solvents, for exampleCHCl₃/McOH, CH₂Cl₂/MeOH, and toluene/MeOH. It may be desirable to warmthe resultant solution to a temperature sufficient to achieve completedissolution. Such a temperature is preferably about 25 to 75° C., morepreferably about 35 to 65° C. After dissolution, undissolved foreignmatter may be removed by hot-filtration or cooling to room temperatureand then filtering. Known methods of filtration may be used (e.g.,gravity filtration, vacuum filtration, or pressure filtration).

(b) The solution is then concentrated to a thick gel/semisolid.Concentration is preferably done by vacuum distillation. Other methodsof concentrating the solution, such as rotary evaporation, may also beused. The temperature of this step is preferably about 20 to 60° C.,more preferably 30 to 50° C.

(c) The thick gel/semisolid is then dispersed in a second blend solvent.The mixture is slurried, preferably near ambient temperature (e.g.,15-30° C.) . Useful second blend solvents are those that cause thephospholipids to precipitate. The second blend solvent is preferablymethyl t-butyl ether (MTBE). Other ethers and alcohols may be used.

(d) The solids produced upon addition of the second blend solvent arethen collected. Preferably the collected solids are washed with anotherportion of the second blend solvent (e.g., MTBE). Collection may beperformed via vacuum filtration or centrifugation, preferably at ambienttemperature., After collection, it is preferred that the solids aredried in vacuo at a temperature of about 20-60° C.

The resultant solid is referred to herein as a phospholipid blend.

Certain of the methods described herein use phospholipids in the form ofa phospholipid blend, including a phospholipid blend made according toany of the blend methods set forth above. Some methods use phospholipidblends, excluding the phospholipid blend made according to themethanol/toluene/MTBE method set forth above wherein a methanol andtoluene mixture is used as the first blend solvent and MTBE is used asthe second blend solvent. For clarity, the method set forth above isreferred to herein as the methanol/toluene/MTBE phospholipid blendmethod.

As used herein, a phospholipid blend is distinguished from otherphospholipid mixtures, including those mixtures that are made fromsimply combining phospholipids in their solid (including powder) forms,as described herein.

In the aqueous suspension-lyophilization methods, phospholipids aresuspended in water at an elevated temperature and then concentrated bylyophilization.

The organic solvent dissolution-precipitation process is preferred overthe aqueous suspension/lyophilization process for a number of reasons asoutlined in U.S. Pat. No. 8,084,056 and published PCT application WO99/36104, including the uniformly distributed phospholipid solid that,results using the organic dissolution method.

Some blend methods that are not the methanol/toluene/MTBE phospholipidblend method recited above use a blend solvent system other thanmethanol and toluene. In these methods, the phospholipids are combinedin a methanol-free and toluene-free condition (also referred to as amethanol and toluene-free condition) to form a phospholipid blend. Thus,a methanol and toluene-free condition refers to a condition that doesnot include both of these solvents.

Some blend methods combine the phospholipids in an blend solvent to formthe phospholipid blend and then evaporate the blend solvent completely,for example either by drying or distillation, to form the driedphospholipid blend. It is this dried phospholipid blend that, is thencontacted with the non-aqueous solvent such as PG in the methodsprovided herein.

Some blend methods combine the phospholipids in an aqueous solvent andthen lyophilize the mixture to form a lyophilized phospholipidcomposition. Other blend methods combine the phospholipids with othersolvent systems such as but not limited to (1) an ethanol andcyclohexane (e.g., 1:1, v:v) mixture, and (2) tertiary butanol(t-butanol or 1,1 dimethyl ethanol), in place of water. Followingdissolution in these various solvents, the compositions are lyophilize.Lyophilization can be performed by freezing over an isopropanol/CO2 bathor an acetone/CO2 bath and drying on a Virtis Lyophilizer until theproduct appears dry and flocculent in appearance.

Various blend methods that are not the methanol/toluene/MTBEphospholipid blend method recited above are described in published EP0923383 (WO1997040858), the entire contents of which are incorporated byreference herein.

Some blend methods that are not the methanol/toluene/MTBE phospholipidblend method recited above combine the phospholipids in a blend solvent,which may be a mixture of toluene and methanol, to form a phospholipidblend, and then precipitate such phospholipid blend in the absence ofMTBE. In these methods, the precipitation occurs in an MTBE-freecondition.

In other blend methods, DPPA, DPPC and MPEG5000-DPPE (or DPPC andMPEG5000-DPPE) may be combined in their dry, solid forms and suchcombination then may be actively and intimately mixed in dry form (e.g.,manually stirring the powders or using a mixing device such as a tumblersilo mixer, an orbiting screw mixer, a ribbon mixer, an extruder, acyclomix, a henschel mixer, a lodige type mixer, an Einch type mixer, orother type of device designed for pharmaceutical powder mixing, with theaim of preparing a uniform blend of phospholipids (e.g., uniformphospholipid dispersion throughout the mixture). Reference can be madeto Deveswaran et al. Research J. Pharm. and Tech.2 (2): April-June 2009)for additional methodologies for generating a uniform dry product.

Non-Blend Methods

In contrast to blend methods, certain non-blend methods involve simplemixing of solid phospholipids and this tends to result in a less uniform(and thus less homogenously dispersed or more heterogeneously dispersed)mixture. These latter mixtures are referred to herein as phospholipidmixtures (or non-blend phospholipid mixtures) in order to distinguishthem from phospholipid blends.

Some ways of preparing phospholipid solutions involve simply contactingphospholipids in their solid forms with the non-aqueous solvent. Thephospholipids may be contacted with the non-aqueous solventsimultaneously or sequentially. If sequentially, any order of additionmay be used. The phospholipids may be added individually to thenon-aqueous solvent or they may be first combined together, in anycombination, and then added to the non-aqueous solvent. Thus, it iscontemplated that the phospholipids may be added to the non-aqueoussolvent individually and simultaneously, individually and sequentially,combined and simultaneously, and partially combined and sequentially. Anexample of the latter is an instance where two of the phospholipids arecombined together in solid form and then contacted with the non-aqueoussolvent before or after the remaining phospholipid is contacted with thenon-aqueous solvent.

Thus, as an example, DPPA, DPPC and MPEG5000-DPPE (or DPPC andMPEG5000-DPPE) phospholipids may be added individually to a non-aqueoussolvent. Such individual addition may be sequential or simultaneousaddition. If sequential, the order of addition can be any order althoughin some instances DPPA may be added second or last since it is the leastabundant and least soluble and its dissolution can be facilitated by thepresence of one of the other phospholipids. In some instances,regardless of whether the phospholipids are provided as individually, oras a simple mixture, or as a phospholipid blend, they are then dissolvedin a non-aqueous solvent comprising PG or a PG/G mixture, as describedabove to form the phospholipid solution. The phospholipid solution maybe combined with gas or it may combined with an aqueous solvent to forma phospholipid suspension which in turn is contacted with gas.

In other instances, a phospholipid blend may be prepared by combiningthe phospholipids in, for example, water, or in an ethanol andcyclohexane (e.g., 1:1, v:v) mixture, or in tertiary butanol (t-butanolor 1,1 dimethyl ethanol), and such mixture is then lyophilized., and thedried product is resuspended in an aqueous solvent. In these instances,the final resuspended product may be combined with a gas such asperflutren. This disclosure contemplates that any or all components usedin this preparation may be analyzed for their divalent metal cationconcentration, and such individual or combined divalent metal cationconcentration may be quantified and used to select and/or prepare a UCA.

Methods of Preparing Ultrasound Contrast Agent, Including Activation

Phospholipid encapsulated gas microspheres are formed by combining andvigorously shaking a phospholipid solution or a phospholipid suspensionswith a gas such as a perfluorocarbon gas. This process is referred toherein as activation. The UCA formulation so formed minimally comprisesphospholipids, non-aqueous solvent such as PG, and gas, and thusactivation minimally results in gas-filled phospholipid microspheres.The phospholipids may be present in an aqueous solution such as is thecase with DEFINITY®, or they may be present in a non-aqueous solutionsuch as is the case with novel UCA formulations including for exampleDEFINITY-II, described in greater detail herein. Thus, in someinstances, activation comprises shaking an aqueous phospholipidsuspension in the presence of a gas, such as a perfluorocarbon gas(e.g., perflutren). In other instances, activation comprises shaking aphospholipid solution in the presence of a gas, a perfluorocarbon gas(e.g., perflutren). It is to he understood that perflutren, perflutrengas and octafluoropropane are used interchangeably herein.

Shaking, as used herein, refers to a motion that agitates a solution,whether aqueous or non-aqueous, such that gas is introduced from thelocal ambient environment within the container (e.g., vial) into thesolution. Any type of motion that agitates the solution and results inthe introduction of gas may be used for the shaking. The shaking must beof sufficient force or rate to allow the formation of foam after aperiod of time. Preferably, the shaking is of sufficient force or ratesuch that foam is formed within a short period of dine, as prescribed bythe particular UCA formulation. Thus in some instances such shakingoccurs far about 30 seconds, or for about 45 seconds, or for about 60seconds, or for about 75 seconds, or for about 90 seconds, or for about120 seconds, including for example for 30 seconds, or for 45 seconds, orfor 60 seconds, or for 75 seconds, or for 90 seconds, or for 120seconds. In some instances, the activation may occur for a period oftime in the range of 60-120 seconds, or in the range of 90-120 seconds.

The disclosure contemplates that, in some instances, the shaking time(or duration) will vary depending on the type of UCA formulation beingactivated. For example, in sonic instances, an aqueous UCA formulationmay be shaken for shorter periods of time than a non-aqueous UCAformulation. The disclosure contemplates that, in such instances, theshaking rate (or shaking speed, as those terms are used interchangeablyherein) may be constant. Thus an activation or shaking means such as anactivation or shaking device may be set to shake at one rate (defined interms of number of shaking motions per minute, for example) for two ormore different pre-determined periods of time.

The disclosure further contemplates that, in some instances, the shakingrate will vary depending on the type of UCA formulation being activated.For example, in some instances, an (aqueous) phospholipid suspension maybe shaken at a slower shaking rate than a (non-aqueous) phospholipidsolution. The disclosure contemplates that, in such instances, theshaking time (or duration, as those terms are used interchangeablyherein) may be constant.

DEFINITY® may be activated with a VIALMIX® as described below. DEFINITY®activation, which involves vigorous shaking of an (aqueous) phospholipidsuspension in the presence of perflutren, lasts for about 45 secondswith a VIALMIX®. Unless indicated otherwise, the term “about” withrespect to activation time intends a time that is +/−20% of the notedtime (i.e., 45+/−9 seconds).

DEFINITY-II may be activated with a VIALMIX® as well. DEFINITY-IIactivation, which involves vigorous shaking of a (non-aqueous)phospholipid solution in the presence of perflutren, lasts for about 60to 120 seconds. In some instances, DEFINITY-II is activated for about 75seconds (i.e., 75+/−15 seconds). DEFINITY-II may be activated for longerperiods of time including 90-120 seconds.

The shaking may be by swirling (such as by vortexing), side-to-side, orup and down motion. Further, different types of motion may be combined.The shaking may occur by shaking the container (e.g., the vial) holdingthe aqueous or non-aqueous phospholipid solution, or by shaking theaqueous or non-aqueous solution within the container (e.g., the vial)without shaking the container (e.g., the vial) itself. Shaking iscarried out by machine in order to standardize the process. Mechanicalshakers are known in the art and their shaking mechanisms or means maybe used in the devices of the present disclosure. Examples includeamalgamators such as those used for dental applications. Vigorousshaking encompasses at least 1000. at least 2000, at least 3000, atleast 4000, at least 4500, at least 5000 or more shaking motions perminute. In some instances, vigorous shaking includes shaking in therange of 4000-4800 shaking motions per minute. VIALMIX® for exampletargets shaking for 4530 “figure of eight” revolutions per minute, andtolerates shaking rates in the range of 4077-4756 revolutions perminute. Vortex* encompasses at least 250, at least 500, at least 750, atleast 1000 or more revolutions per minute. Vortexing at a rate of atleast 1000 revolutions per minute is an example of vigorous shaking, andis more preferred in some instances. Vortexing at 1800 revolutions perminute is most preferred.

The shaking rate can influence the shaking duration needed. A fastershaking rate will tend to shorten the duration of shaking time needed toachieve optimal microbubble formation. For example, shaking at 4530 rpmfor a 45 second duration will achieve 3398 total revolutions on aVIALMIX®. Shaking at 3000 rpm would require 68 seconds to achieve thesame number is of revolutions. The duration and shake speed requiredwill also be influenced by the shape of the travel path and amplitude ofshaking. The velocity the liquid in the container reaches and the forcesexerted upon change of direction will influence gas incorporation. Theseaspects will be impacted upon based on the shaker arm length and path,the container shape and size, the fill volume and the formulationviscosity. Water has a viscosity of approximately 1.14 cps at 15° C.,(Khattab, L S, et al., Density, viscosity, surface tension, and molarvolume of propylene glycol+water mixtures from 293 to 323 K andcorrelations by the Jouyban-Acree model Arabian Journal of Chemistry(2012). In contrast, propylene glycol has a viscosity of 42 cps at 25°C. (Khattab, I. S. et al., Density, viscosity, surface tension, andmolar volume of propylene glycol+water mixtures from 293 to 323 K andcorrelations by the Jouyban-Acree model Arabian Journal of Chemistry(2012) and glycerol has a viscosity of 2200 cps at 15° C. (Secut J B,Oberstak H E Viscosity of Glycerol and Its Aqueous Solutions. Industrialand Engineering Chemistry 43. 9 2117-2120 1951). DEFINITY-II has a highviscosity of 1150cps at 15° C. Since DEFINITY® is predominantly water ithas a much lower viscosity than DENNITY-II.

The formation of gas-filled microspheres upon activation can be detectedby the. presence of a foam on the top of the aqueous or non-aqueoussolution and the solution becoming white.

Activation is carried out at a temperature below the gel state to liquidcrystalline state phase transition temperature of the phospholipidemployed. By “gel state to liquid crystalline state phase transitiontemperature”, it is meant the temperature at which a phospholipid layer(such as a lipid monolayer or bilayer) will convert from a gel state toa liquid crystalline state. This transition is described for example inChapman et al., J. Biol. Chem. 1974 249, 2512-2521. The gel state toliquid crystalline state phase transition temperatures of variousphospholipids will be readily apparent to those skilled in the art andare described, for example, in Gregoriadis, ed., Liposome Technology,Vol. 1, 1-18 (CRC Press, 1984) and Derek Marsh, CRC Handbook of LipidBilayers (CRC Press, Boca Raton, Fla. 1990), at p. 139. Vigorous shakingcan cause heating of the formulation based on the shake speed, duration,shaker arm length and path, the container shape and size, the fillvolume and the formulation viscosity.

It will be understood by one skilled in the art, in view of the presentdisclosure, that the phospholipids car phospholipid microspheres may bemanipulated prior to or subsequent to being subjected to the methodsprovided herein. For example, after the shaking is completed, thegas-filled microspheres may be extracted from their container (e.g.,vial). Extraction may be accomplished by insetting a needle of a syringeor a needle-free spike (e.g., PINSYNC®) into the container, includinginto the foam if appropriate, and drawing a pre-determined amount ofliquid into the barrel of the syringe by withdrawing the plunger or byadding an aqueous liquid, mixing and drawing a pre-determined amount ofliquid into the barrel of the syringe by withdrawing the plunger. Asanother example, the gas-filled microspheres may be filtered to obtainmicrospheres of a substantially uniform size. The filtration assemblymay contain more than one filter which may or may not be immediatelyadjacent to each other.

Methods of Using Ultrasound Contrast Agent to Image a Subject

Also provided herein are methods of use of phospholipid-encapsulated gasmicrospheres and formulations thereof. The gas microspheres andformulations thereof may be used in vivo in human or non-human subjects,or they may be used in vitro. They may be used for diagnostic ortherapeutic purposes or for combined diagnostic and therapeuticpurposes.

When used in human subjects, phospholipid-encapsulated gas microspheresand formulations thereof may be used directly (neat) or may be dilutedfurther in a solution, including a pharmaceutically acceptable solution,and administered in one or more bolus injections or by a continuousinfusion. Administration is typically intravenous injection, Imaging isthen performed shortly thereafter. The :imaging application can bedirected to the heart or it may involve another region of the body thatis susceptible to ultrasound imaging. Imaging may be imaging of one ormore organs or regions of the body including without limitation theheart, blood vessels, the cardiovasculature, the liver, the kidneys andthe head.

Subjects of the invention include but are not limited to humans andanimals. Humans are preferred in some instances. Animals includecompanion animals such as dogs and cats, and agricultural or prizeanimals such as but not limited to hulls and horses.

UCAs are administered in effective amounts. An effective amount will bethat amount that facilitates or brings about the intended in vivoresponse and/or application. In the context of an imaging application,such as an ultrasound application, the effective amount may be an amountof phospholipid-encapsulated gas microspheres that allow imaging of asubject or a region of a subject.

EXAMPLES 1 Examples Methods 1.1 Phospholipids and Phospholipid Blendsand Reagents

Phospholipids were used as either individual powders, combined togetheras powders and used as a mixture or blended together by dissolving anddrying (details described below). The measured content of the individualphospholipids was used to estimate the final calcium or magnesiumconcentrations in the non-aqueous concentrate or the aqueous preparationunless direct measurements in the blend was made. Solvents with lowcalcium were used for all studies.

1.1.1 Phospholipid Blend

One phospholipid blend (LB) was prepared by dissolving DPPC, DPPA,MPEG5000-DPPE (0.401:0.045:0.304 [wt:wt:wt]) in toluene/methanol,concentrated with vacuum and warming and then slurried by the additionof Methyl t-butyl ether (MTBE). The solid material was collected, washedwith MTBE and dried (consistent with patent U.S. Pat. No. 8,084,056),Alternatively, DPPC, DPPA, and MPEG5000-DPPE (0.401:0.045:0.304[wt:wt:wt]) were solubilized at 55° C. in methanol. The methanol wasthen evaporated and the solids recovered as phospholipid blend.Similarly, DPPC, DPPA, and MPEG5000-DPPE (0.401:0.045:0.304 [wt:wt:wt])were combined together as solid powders and the powders were mixedtogether with a spatula.

1.1.1.1 Residual Solvent Method for Phospholipid Blend

Residual solvent in phospholipid blend was determined by FID using GCheadspace. Sample was weighed, transferred into a separate 20 ccheadspace vials and dissolved in N-methylpyrrolidone. A set of residualsolvent standards was prepared in N-methylpyrrolidone. Standards andsamples were analyzed by FID using GC headspace. The concentration ofeach residual solvent was calculated from the calibration curve for thatsolvent.

1.1.2 Calcium Measurements

Calcium levels were quantified in individual lipid, lipid blend,glycerol and propylene glycol using either ICP-MS or AA, Magnesium andother metal ions were also measured with these methods in some samples

1.1.1.2 ICP-MS (Inductively Coupled Plasma—Mass Spectrometry) Method

Samples were prepared by weighing into a pre-cleaned quartz digestionvessel. Matrix spikes were added and then mixed with nitric acid andhydrochloric acid. The samples were digested in a closed-vesselmicrowave digestion system. After cooling internal standard solutionwere added and diluted and analyzed by ICP-MS using He collision mode.

1.1.1.3 AA (Atomic Absorption Spectroscopy) Method

Samples were prepared by weighing into a dry “trace metals cleaned”digestion vessel and dissolved with nitric acid and hydrochloric acidand reflux with H₂O₂. The sample solution was washed with water andfiltered. A set of standards were used to calibrate the AA and then theabsorbance of the samples read from the calibration curve. Results forindividual lipid, phospholipid blend, and formulations solvents areprovided in Table 1.

TABLE 1 Ca⁺2 and Mg+2 Level in Individual Lipid, Lipid Blend and Solvent^(a) Materials Ca⁺² (ppm) ^(b) Mg⁺² (ppm) ^(b) Phospholipid Blend, Lot 1^(c) Not detected Not detected Phospholipid Blend Lot 2 ^(c, d) 370  54MPEG5000-DPPE (high Ca⁺²) Lot 1 980 150 MPEG5000-DPPE (high Ca⁺²) Lot 2520 110 MPEG5000-DPPE (low Ca⁺²) 4 Not determined DPPC Lot 1 Notdetected Not detected DPPC Lot 2 7 Not determined DPPA 19 Not determinedPropylene glycol Not detected Not determined Glycerol 0.7 Not determined^(a) Determined by ICP-MS ^(b) ppm = parts per million and is equivalentto μg/g ^(c) Phospholipid blend consists of DPPC, DPPA and MPEG5000-DPPE(0.401:0.045:0.304 [wt:wt:wt]) ^(d) Lipid blend, Lot 2 prepared usingMPEG5000-DPPE (high Ca⁺²) Lot 1

1.2 Aqueous Formulation Preparation 1.2.1 Non-Aqueous PhospholipidConcentrate:

Phospholipid concentrates were prepared by adding the individual lipids(DPPC, DPPA, and MPEG5000-DPPE low Ca⁺², or MPEG5000-DPPE high Ca⁺², ora combination) in any order, adding phospholipid blend (LB), or addingLB containing high levels of Ca⁺² to 25-115 mL of propylene glycol (PG),or 1:1 v/v propylene glycol/glycerol (PG/G), or glycerol vehicle withconstant stirring at 55° C. to 70° C. In some cases, lipid concentratewas prepared without DPPA or with calcium acetate added prior to lipidaddition.

1.2.2 Aqueous Formulation:

Aqueous formulations were prepared by adding: dibasic sodium phosphate,heptahydrate; monobasic sodium phosphate, monohydrate; sodium chloride;propylene glycol; glycerol and finally non-aqueous phospholipidconcentrate to 400 to 500 mL of water with constant stirring at 55° C.to 70° C. In some cases, calcium acetate was added prior to, or afteraddition of the non-aqueous phospholipid concentrate to the hulkcompounding solution. In other cases the phosphate buffer was notincluded.

1.3 Non-Aqueous Formulation Preparation 1.3.1 Non-Aqueous Formulation

Individual lipids (DPPC, DPPA, and MPEG5000-DPPE low Ca⁺² orMPEG5000-DPPE high Ca⁺², or a combination of both) in any order, LB, orLB containing high levels of Ca⁺² were added to 25 to 100 mL ofpropylene glycol (PG) containing 0.005 M acetate buffer (90/10, sodiumacetate/glacial acetic acid) vehicle with constant stirring at 60° C.±5°C. Following dissolution, glycerol was then added to produce thenon-aqueous formulation.

1.4 Calcium and/or Magnesium Additions Using Stock Solutions

1.4.1 Initial Studies

Stock solutions of calcium acetate, magnesium acetate alone and amixture of both were prepared in propylene glycol (25.4 μg, Ca⁺²/g, 28.0μg Mg⁺²/g and 14.0 μg Ca⁺²/g with 12.7 μg Mg⁻²/g respectively). Theindividual stock solutions were added in aliquots up to a total of 1 mLin 33 mL of propylene glycol containing lipid blend (15 mg/mL). Thesolutions were compared to propylene glycol alone, and the solutionfirst showing cloudiness recorded.

1.4.2 Follow-Up Studies with Reference Scale

Stock solutions of calcium acetate, monohydrate were prepared inpropylene glycol (299 Ca⁺² μg/g), propylene glycol and glycerol (299Ca⁺² μg/g), or water (6085 Ca⁺² μg/g) and vehicle matched when added tothe propylene glycol, the non-aqueous phospholipid concentrate or theaqueous formulation (before or after addition of the non-aqueousphospholipid concentrate). The maximum added calcium stock was always<12% of the total volume.

Some non-aqueous phospholipid concentrates were titrated with calciumacetate. The appearance was evaluated on a 0, +, ++, +++ scale by visualinspection. FIG. 1 provides the scale used for the determinations, andwas generated using low Ca⁺² lipids (DPPC, DPPA and MPEG5000-DPPE;0.401:0.045:0.304 [wt:wt:wt]) formulated in propylene glycol at 15 mgtotal lipid/mL.

1.5 Filtration: 1.5.1 Aqueous Formulation

Prepared samples of phospholipid aqueous suspensions were held at 55° C.prior to filtration. Samples were placed in a 55° C. temperaturecontrolled 60 mL syringe with a 13 mm Hydrophilic PolyvinylideneFluoride (PVDF) 0.22 am membrane syringe filter attached. A 5 psinitrogen head pressure was applied to the syringe. Flow rate wasdetermined by weighing the filtered solution over time with readingsevery 30 seconds. Flow rates per time point were calculated and theaverage flow between 9 to 10 minutes was compared to the initial flow (0to 1 minute) and expressed as a percentage. A pre-filtration sample wascollected along with to samples throughout the filtration forphospholipid concentration analysis.

1.5.2 Non-Aqueous Formulation

Prepared samples of phospholipid non-aqueous solutions were held at 60°C. prior to filtration. Samples were placed in a 60° C. temperaturecontrolled 60 mL syringe with 25 mm Hydrophilic Polyethersulfone (PES)0.2 μm membrane syringe filter attached. A 10 psi nitrogen head pressurewas applied to the syringe. Flow rate was determined by weighing thefiltered solution over time with readings every 30 seconds. Flow ratesper time point were calculated and the average flow between 8 to 9minutes was compared to the initial flow (0 to 1 minute) and expressedas a percentage. Flow rates in clear solutions were seen to increasewith time as the filter warmed. Samples were collected as outlinedabove.

1 Phospholipid Assay:

In some cases samples were assayed for phospholipid content. The samplewas transferred to a HPLC vial and analyzed by reverse phase HPLCseparation and Corona Charged aerosol detection. (CAD; HPLC With ChargedAerosol Detection for the Measurement of Different Lipid Classes, I. N.Acworth, P. H. Gamache, R, McCarthy and D. Asa, ESA Biosciences Inc.,Chelmsford, Mass., USA; J. Waraska and I. N. Acworth, AmericanBiotechnology Laboratory, January 2008) and quantified versus referencestandards.

1.7 Product Preparation and Testing 1.7.1 Aqueous Formulation

Filtered aqueous formulation (see section 1.5.1) was aliquoted (1.76 mL)into 2 cc Wheaton vials the headspace air replaced with petfluoropropane(PFP) gas, the vial sealed with a West grey butyl stopper, and crimpedwith an aluminum seal.

1.7.2 Non-Aqueous Formulation

Filtered aqueous formulation (see section 1.5.2) was aliquoted (0.35 mL)into 2 cc Wheaton vials the headspace air replaced with petfluoropropane(PFP) gas, the vial sealed with a West grey butyl stopper, and crimpedwith an aluminum seal.

1.7.3 Sysmex Microsphere Sizing:

Samples were analyzed for number and size distribution using a particlesizer (Malvern FPIA-3000 Sysmex). Aqueous or non-aqueous samples wereoptimally activated using a VIALIMX®, a portion of the activated productdiluted with saline and then transferred to the sample vessel of theSysmex. The Sysmex uses an appropriate sheath solution and analyzes thesample using both low and high power fields to generate sizing data forthe specified size range (1 to 80 μm in the current studies).

1.7.4 Ultrasound Contrast of Activated Product:

Acoustic attenuation was measured for selected samples using a PhilipsSonos 5500 clinical ultrasound imaging system. Following optimalactivation with a VIALMIX® 10 microliter samples were pipetted into a250 mL beaker containing 200 ml of 0.9% saline at room temperature. Around, vaned, 38 mm diameter stirring bar maintained solution uniformityand served as an acoustic reflector. The s3 clinical transducer of theultrasound system was positioned at the top of the beaker, just into thesolution and 4.8 cm above the upper margin of the stirring bar. Fiveseconds of 120 Hz images were then acquired digitally and written todisk beginning 10 seconds after introduction of the sample. The USsystem was used in IBS mode, TGC, was fixed at the minimal value for alldepths, and LGC was disabled. The mechanical index (MI) was 0.2 withpower set 18 dB below maximum. The receive gain was fixed at 90 and thecompression at 0. For each sample tested, US data acquisition wasacquired prior to (blank) and after sample injection.

Image analysis was performed using Philips QLab version 2.0, which readfiles produced by the US system and calculated values in dB for IBSmode. Regions of interest were drawn on the stirring bar and the dBvalues exported to Excel. These were then averaged over the full 5second (approximately 360 video frame) acquisition. Attenuationmeasurements were obtained by subtracting the averaged sample ROI valuefrom the averaged blank ROI value (both in dB). This was divided bytwice the distance between the US transducer and the upper margin of thestiffing bar to yield attenuation in dB/cm. Values were then divided bythe calculated microbubble concentration in the beaker and expressed interms of dB attenuation per centimeter per million microbubbles/mL.

Example 1 Effect of Calcium Addition to Non-Aqueous PhospholipidSolution

This example demonstrates the effect of calcium and magnesium ions onphospholipid precipitation.

Example 1.1 Initial Studies on the Effect of Calcium and MagnesiumAddition to Non-Aqueous Solution

In initial studies, lipid blend (LB, Lot 1) characterized as having lowdivalent metal ion concentration (Table 1 in example methods), was addedto propylene glycol at 55±5° C. and stirred. It was verified by visualobservation that the phospholipids had fully dissolved and the resultingsolution was clear. This LB solution was titrated with calcium (25.4 μgCa⁺²/g), magnesium (28.0 μg Mg⁺²/g) or a combination (1:1 to make asolution containing 14.0 μg Ca⁺²/g and 12.7 μg Mg⁺²/g) and showedcloudiness at 3.60 μg Ca⁺²/g, 4.23 μg Mg⁺²/g and 2.35 μg/g combinedmetal ion 1g non-aqueous phospholipid solution, respectively.

Example 1.2 Follow-Up Studies on the Effect of Calcium Addition toNon-Aqueous Solution

The experiment was conducted as follows: DPPC, DPPA and MPEG5000-DPPFpowder, characterized as having low divalent metal ion concentration(Table 1 in example methods), were added either individually (in thesequence shown in Table 2) or as a mixture or as a blend added to heated(55° C.±5° C.) and stirred propylene glycol (PG) or a 1:1 mixture ofpropylene glycol and glycerol (PG/G). It was verified by visualobservation that the phospholipids had fully dissolved and the resultingsolution was clear (=0: see example methods Section 1.4.2, FIG. 1 forrating scale). FIG. 2 illustrates the appearance of a lipid concentratein propylene glycol upon the successive additions of DPPC,MPEG5000-DPPE, DPPA and calcium acetate stock (1 mL, of a 299 μg Ca⁺²per mL of stock was added t© produce a lipid concentrate with an 11.1 μgCa⁺² per g of solution). The lipid concentrate did not turn cloudy untilthe calcium was added.

Phospholipid solutions in PG or 1:1 mixture PG/G were titrated by aseries of small additions of calcium. After each addition, the solutionwas assessed for clarity (see example methods Section 1.4.2, FIG. 1 forrating scale) and the lowest calcium concentration producing a +, ++,+++ and score is shown in Table 2. FIG. 3 shows representative solutionsfor the Study 4 titration.

TABLE 2 Effect of Calcium addition to Non-aqueous phospholipid solutionObserved cloudiness Order of lipid addition Non- thresholds (μg Ca⁺²/g)for MPEG5000- aqueous titration with calcium ^(a, b) Study DPPC DPPEDPPA Solvent + ++ +++ 1 ^(c) 1 3 2 PG 1.5 2.6 >5.7 2 ^(c) 2 3 1 PG 1.52.9 >11.1 3 ^(c) 3 1 2 PG 2.3 4.6 11.1 4 ^(c) 1 2 3 PG 1.8 2.9 >5.7 5^(d) phospholipid Blend PG 1.8 5.7 11.1 6 ^(e) phospholipid Mixture(dry) PG 1.8 5.7 11.1 7 ^(f)   1 3 2 PG & G Lipids not dissolved 8 ^(g)1 3 2 PG & G 2.6 19.2 35.8 ^(a) Defined in methods Section 1.4 ^(b)Titrated with calcium acetate stock solutions (299 μg Ca⁺²/g of stocksolution) ^(c) DPPA (0.9 mg/mL), DPPC (8.02 mg/mL) and MPEG5000-DPPE(6.08 mg/mL) final concentration was achieved by individual phospholipidaddition to propylene glycol (25 mL) ^(d) Phospholipid blend (15 mg/mL),made using methanol to dissolve phospholipids at 55° C. followed bydrying, dissolved in 25 mL propylene glycol ^(e) Phospholipid mixture:DPPA, DPPC, and MPEG5000-DPPE (0.045:0.401:0.304), powders were stirredtogether and used for compounding in 25 mL propylene glycol. The finalconcentration is 15 mg/mL ^(f) Phospholipid solution made by addingindividual phospholipids [DPPA (0.9 mg/mL), DPPC (8.02 mg/mL) andMPEG5000-DPPE (6.08 mg/mL)] to 25 mL, 1:1 (v/v) propyleneglycol:glycerol ^(g) Phospholipid solution made by adding individualphospholipids [DPPA (0.225 mg/mL), DPPC (2.00 mg/mL) and MPEG5000-DPPE(1.70 mg/mL)] to 100 mL, 1:1 (v/v) propylene glycol:glycerol

Calcium titration produced a clear concentration dependent precipitationin the phospholipid solution irrespective of the how the phospholipidswere added (individually, as a mixture or as a blend) to the propyleneglycol (see Table 2). The lipids were not soluble in either glycerolalone or in 25 mL of 1:1 PG/G but did achieve a clear solution whenadded to 100 mL of 1:1 PG/G (Study 8). Calcium produced a concentrationdependent precipitation in this lipid solution consistent with initialfindings (see Table 2). Overall, these titration studies indicated thelowest calcium, magnesium and combined concentrations that producedprecipitation was 1.5 μg Ca⁺²/g, 4.23 μg Mg⁺²/g, and 2.35 μg combinedmetal ion/g non-aqueous phospholipid solution.

Example 2 Effect of Phospholipid Solution Components Containing Calciumwhen Mixed Example 2.1 Calcium in PG

Study 9 was conducted as follows: DPPC, MPEG5000-DPPE and DPPA powder,characterized as having low calcium concentration (see Table 1 inexample methods), were added individually (in the sequence shown inTable 3) to heated (55° C.±5° C.). and stirred PG containing 11 μg/gcalcium. Clarity was assessed (see Section 1.4) and the solution wasclear after DPPC dissolved, turned and stayed cloudy after addition ofDPPA, and remained cloudy after addition of MPEG5000-DPPE. Thecloudiness observed was scored as +++ (FIG. 1, Section 1.4). Thiscontrasted with the clear solution produced when these phospholipids(including DPPA) were added to PG containing low calcium (startingsolution for Study 1). This was further emphasized by Study 12, whereonly phospholipids DPPC and MPEG5000-DPPE containing high Ca⁺² levels,were dissolved and this solution stayed clear even with the presence ofcalcium.

Example 2.2 Calcium in Lipid Blend from MPEG5000-DPPE

Initial experiments were performed on phospholipid blend (made usingtoluene and methanol to dissolve and adding MTBE to precipitate out thelipid blend) containing DPPC, DPPA and either low (not detected Ca⁺² and1 μg Mg⁺²/g, MPEG5000-DPPE) or high (980 μg Ca⁺²/g and 150 μg Mg⁺²/g,MPEG5000-DPPE Lot 1) calcium and magnesium containing MPEG5000-DPPE,respectively, were added to heated (55° C.±5° C.) and stirred propyleneglycol. The two lipid blends were mixed to provide samples havingapproximately 0, 1.75, 4.11 and 12.9 μg combined Ca⁺² & Mg⁺²/g ofnon-aqueous phospholipid solution. The 1.75 μg combined Ca⁺²/g Mg⁺²/g ofnon-aqueous phospholipid solution showed cloudiness.

Follow-up study 10 and 11 were conducted as follows: phospholipid blend(made using toluene and methanol to dissolve and adding MTBE toprecipitate out the phospholipid blend) containing DPPC, IPPA and eitherhigh (980 ppm Ca⁺², 150 ppm Mg⁺², Lot 1) or low (4 ppm Ca⁺²) calciumcontaining MPEG5000-DPPE were added to heated (55° C.±5° C.). andstirred PG, Clarity was assessed (see Section 1.4) and slight cloudinesswas observed (+; see example methods in Section 1.4) with thephospholipid blend containing high calcium (measured as 370 ppm Ca⁺² and54 ppm Mg⁺²). This contrasted with the clear solution produced bydissolving low calcium (non-detectable levels of Ca⁺² and Mg⁺²)containing phospholipid blend (see Table 3).

TABLE 3 Effect of Phospholipid solution components containing calciumwhen mixed Ob- served Order of phospholipid addition Non- Cloudi-MPEG5000- aqueous Ca⁺² (Mg⁺²) ness Study DPPC DPPE DPPA Solvent μg/g[source] Level ^(a)   9 ^(b) 1 2 3 PG 11.2 (0.0)   +++ ^(c) [added to PGbefore lipid addition]   12 ^(d) 1 2 — PG 5.8 (0.9)   0 ^(e) [High Ca⁺²MPEG5000- DPPE] 10 Phospholipid blend PG 0.0 (0.0) 0 containing low Ca⁺²^(f) [LB Lot 1] 11 Phospholipid blend PG 5.36 (0.8) +++ containing highCa⁺² ^(f) [LB Lot 2] ^(a) Defined in methods Section 1.4 ^(b) DPPA (0.9mg/mL), DPPC (8.02 mg/mL) and MPEG5000-DPPE (6.08 mg/mL) finalconcentration was achieved by individual phospholipid addition topropylene glycol (25 mL) ^(c) Solution was clear when DPPC solubilized,remained clear after addition of MPEG5000-DPPE turned cloudy afteraddition of DPPA ^(d) DPPC (8.02 mg/mL) and MPEG5000-DPPE containingCa⁺² (6.08 mg/mL; 980 ppm Ca⁺² and 150 ppm Mg⁺²); final concentrationwas achieved by individual phospholipid addition to propylene glycol (25mL), no DPPA added ^(e) Solution was clear upon addition of DPPC andMPEG5000-DPPE ^(f) Phospholipid blend (15 mg/mL) made using toluene andmethanol to dissolve phospholipids and adding MTBE to precipitate outthe phospholipid blend, dissolved in 25 mL propylene glycol

Example 2.3 Calcium from MPEG5000-DPPE Added Individually

Studies 13 through 17 were conducted as follows: DPPA and DPPC,characterized as having low calcium concentration (see Table 1 inexample methods:), were added individually (in the sequence shown inTable 4) to heated (55° C.±5° C.). and stirred PG. MPEG5000-DPPEcontaining different proportions of “low” and “high” calcium andmagnesium material was added. Clarity was assessed (see example methodsin Section 1.4, FIG. 1) and a calcium and magnesium concentrationdependent precipitation was observed (see Table 4 and FIG. 4)

TABLE 4 Calcium and Magnesium from MPEG5000-DPPE added as an individualcomponent Percentage ^(c) Metal ion Order of MPEG5000- MPEG5000- Non-Concentration Observed lipid addition DPPE DPPE aqueous (μg/g)Cloudiness Study DPPC DPPA (Low Ca⁺²) (high Ca⁺²⁾ Solvent Ca⁺² Mg⁺²Total Level ^(a) 13 ^(b) 1 2 100 0 PG 0.1 0.0 0.1 0 14 ^(b) 1 2 75 25 PG0.7 0.1 0.8 + 15 ^(b) 1 2 50 50 PG 1.3 0.3 1.6 ++ 16 ^(b) 1 2 25 75 PG1.9 0.4 2.3 ++ 17 ^(b) 1 2 0 100 PG 3.1 0.6 3.7 +++ 18   1 2 n/p ^(d)n/p ^(d) PG & G ^(e) Not added Cloudy, DPPA not dissolved ^(a) Definedin methods Section 1.4 ^(b) DPPA (0.9 mg/mL), DPPC (8.02 mg/mL) andMPEG5000-DPPE (6.08 mg/mL) final concentration was achieved byindividual phospholipid addition to propylene glycol (25 mL) ^(c)Percentages of MPEG5000-DPPE; low Ca⁺² [4 ppm] and high Ca⁺² [520 ppmCa⁺², 110 ppm Mg⁺²] relative to total ^(d) n/p = not performed;Phospholipids [DPPC (8.02 mg/mL) and DPPA (0.9 mg/mL)] not solubilizedin propylene & glycol solvent system ^(e) Propylene glycol and glycerol50:50 (v/v)

Summary of Example 2

Overall these studies have demonstrated the addition of calcium, eitherin the non-aqueous solvent or via the phospholipid blend or when addedas MPEG5000-DPPE as an individual compound, all caused precipitation.The concentration where effects were seen were similar for Example 2compared to those in Example 1. The lowest calcium concentration thatproduced cloudiness (+) was at 0.7 μg/g Ca⁺² (0.8 μg/g total Ca⁺² andMg⁺²). This is a concentration to the 1.5 to 2.6 μg/g see in Example 1.

Example 3 Addition of Non-Aqueous phospholipid Solution to AqueousSolvent Example 3.1 Effect of Calcium in Non-Aqueous phospholipidSolution on Addition to Aqueous Solvent

A series of studies were performed to examine the impact of calcium inthe non-aqueous phospholipid solution prior to transferring into theaqueous formulation. These involved the steps of: 1) preparing anon-aqueous phospholipid solution, 2) preparing an aqueous solution and3) combining solutions from 1 and 2.

Example 3.1.1 Preparing Non-Aqueous Solution: Calcium Added toNon-Aqueous Solution After phospholipids Dissolved

Consistent with Example 2, the first step in studies 19, 20 and 22 wereas follows: DPPC, DPPA, and MPEG5000-DPPE powder, characterized ashaving low calcium concentration (see Table 1 in example methods), wereadded individually (in the sequence shown in Table 5) to heated (55°C.±5° C., with the exception of study 22, which was heated to 70° C.)and stirred propylene glycol. It was verified by visual observation thatthe phospholipids had fully dissolved and the resulting solution wasclear (see example. methods in Section 1.4, FIG. 1). A solution ofcalcium acetate [Ca(OAc)₂] in propylene glycol was added as indicated inTable 5, the solution was stirred and observed for changes inappearance, as compared to a solvent blank and the assessment of claritywas recorded. Upon addition of calcium acetate, the solutions turnedcloudy. These propylene glycol concentrates were transferred to theaqueous phase as described below.

Example 3.1.2 Preparing Non-Aqueous Solution: Calcium in MPEG5000-DPPE

The first step studies 21 and 25 were as follows: DPPC, DPPA (notincluded in study 25). and calcium containing MPEG5000-DPPE powder (980ppm,114PEG5000-DPPE Lot 1; see Table 1 in example methods), were addedindividually (in the sequence shown in Table 5) to heated (55° C.±5° C.)and stirred in PG. Clarity was assessed and significant cloudinessobserved in study 21 (+++; see example methods in Section 1.4, FIG. 1)after addition of DPPA, and remained cloudy after addition ofMPEG5000-DPPE, whereas no cloudiness was observed in study 25 which didnot contain DPPA. These non-aqueous phospholipid solutions weretransferred to the aqueous phase as described below.

Example 3.1.3 Preparing Non-Aqueous Solution: Calcium in Lipid Blendfrom MPEG5000-DPPE

Consistent with Example 2. the first step in studies 23 and 24 wereconducted as follows: phospholipid blend (made using toluene andmethanol to dissolve and adding MTBE to precipitate out the lipid blend)containing DPPC, DPPA and either low (4 ppm. lot 2 or high (980 ppm,MPEG5000-DPPE Lot 1) calcium containing MPEG5000-DPPE, respectively,were added to heated (55° C.±5° C.) and stirred propylene glycol.Clarity was assessed and significant cloudiness observed (+++; seeexample methods in Section 1.4, FIG. 1) with the phospholipid blendcontaining high calcium. This contrasted with the clear solutionproduced by dissolving low calcium containing phospholipid blend (seeTable 5). These non-aqueous phospholipid solutions were transferred tothe aqueous phase as described below.

Example 3.1.4 Preparing Non-Aqueous Solution: Calcium in PG Prior toAdding phospholipids

Consistent with Example 2, the first step in studies 28 and 30 wereconducted as follows: DPPC, MPEG5000-DPPE and DPPA powder, characterizedas having low calcium concentration (see Table 1 in example methods),were added individually (in the sequence shown in Table 5) to heated(55° C.±3° C.). and stirred PG either containing 11 μg/g calcium orcalcium added after phospholipid addition, respectively. Clarity wasassessed (see example methods in Section 1.4, FIG. 1) and in study 2.8the solution was clear after DPPC and MPEG5000-DPPE dissolved but turnedand stayed cloudy after addition of DPPA. In study 30 the solution wasclear after DPPC, DPPA and MPEG5000-DPPE were dissolved, and turnedcloudy after addition of Ca⁺². The cloudiness for both studies wasscored as +++ (see Table 5). These non-aqueous phospholipid solutionswere transferred to the aqueous phase as described. below.

Example 3.2 Preparing Aqueous Solution

For all studies the aqueous solution was prepared as follows: In aseparate vessel Sodium Chloride (NaCl), Sodium Phosphate DibasicHeptahydrate (Na₂HPO₄.7H₂O), and Sodium Phosphate Monobasic(NaH₂PO₄.H₂O) were added to water in a stirred vessel, and mixed untildissolved. Propylene glycol and glycerol were also added, as needed, sothe final addition of phospholipid concentrate will reconstitute to an8:1:1 water: glycerol: propylene glycol composition. This stirredsolution was maintained at 55° C.±5° C. (with the exception of study 22where the aqueous solution was maintained at 70° C.).

Example 3.3 Combining Non-Aqueous and Aqueous Solutions

For all studies, the addition of the non-aqueous phospholipidconcentrate to the aqueous solution was done as follows: The warmphospholipids in propylene glycol were added and stirred at 100 to 150rpm. Visual observations were recorded and the time for lull dispersionor dissolution was (either clear or Cloudy) noted. These aqueoussuspensions were then collected and filtered through a 0.2 um filter at55° C. under 5 psi head pressure. Flow rate was measured and samplescollected for phospholipid measurement (see example methods forprocedure). Pre- and post-filtration samples were assayed to determinethe level of phospholipid loss associated with filtration.

TABLE 5 Effect of divalent metal ion in non-aqueous phospholipidsolution on addition to aqueous solvent Aqueous suspension ^(b) Percentof Initial Non-aqueous phospholipid concentrate ^(a) Ca⁺² (Mg⁺²)Filtration % phospholipid Phospholipid Ca⁺² (Mg⁺²) Contains Appearanceconcentration Rate at post filtration ^(d) addition μg/g PG Observedphosphate after addition in aqueous 9 to 10 MPEG5000- Study to PG [Ca⁺²source] cloudiness ^(c) buffer to aqueous [μg/g water] minutes DPPC DPPADPPE 19 C, E, A 0.0 (0.0) 0 Yes Clear  0 (0) 64.6 101 100 99 20 C, E, A13.7 (0)^(e) +++ Yes Cloudy 0.8 (0) 1.3; 95 76 94 [Calcium blockedacetate added filter after lipids] 21 C, A, E 3.1 (0.7) ^(f) +++ YesCloudy   0.2 (0.03) 9.0; 96 78 95 [in MPEG5000- blocked DPPE] filter  22 ^(g) C, A, E 21.4 (0)^(e) +++ Yes; at Cloudy 1.2 (0) 8.5; 98 75 97[Calcium 70° C. blocked acetate added filter after lipids] 25 C, E 5.8(0.9)^(h) 0 Yes Clear   0.3 (0.04) 82.1 100 nd 99 [in MPEG5000- DPPE] 23LB ^(i) 0 (0) 0 Yes Clear  0 (0) 92.0 99 100 99 24 LB ^(i) 5.36 (0.8)+++ Yes Slightly   0.3 (0.04) 5.2 98 65 96 [Lipid Blend] cloudy blockedfilter 28 C, E, A 11.2 (0.0) ^(k) +++ Yes Cloudy 0.6 (0) 42.7 101 24 100[Calcium acetate in PG, then phospholipid added] 30 C, A, E 21.4(0.0)^(e) +++ No Cloudy 1.2 (0) 9.0 89 42 86 [Calcium blocked acetateadded filter after lipids] ^(a) Phospholipid concentrate was prepared at15 mg/mL by dissolving DPPC (C), MPEG5000-DPPE (E) and DPPA (A) in theratio of 0.401:0.304:0.045 in propylene glycol in the order listed at55° C. ^(b) Phospholipid concentrates were added to a compounding vesselcontaining: water (800 mg); dibasic sodium phosphate, heptahydrate (2.16mg); monobasic sodium phosphate, monohydrate (2.34 mg); sodium chloride(4.84 mg), glycerol (126 mg), and propylene glycol (51.75 mg) per mL ofcompounding solution. Materials were combined at 55° C., in the orderlisted. ^(c) Defined in methods Section 1.4.2 ^(d) HPLC with CADdetection described in Section 1.6 ^(e)1 mL, 2 mL and 2 mL (Study 20,22, and 30, respectively) of a stock 299 μg Ca⁺² per g PG, after lipidaddition prior to transfer to the aqueous compounding solution ^(f)MPEG5000-DPPE containing Ca⁺² (6.08 mg/mL; 520 and 110 ppm Ca⁺² andMg⁺²) used for experiment ^(g) All compounding performed at 70° C.^(h)MPEG5000-DPPE containing Ca⁺² (6.08 mg/mL; 980 and 150 ppm Ca⁺² andMg⁺²) used for experiment ^(i) Made using toluene and methanol todissolve lipids and adding MTBE to precipitate out the lipid blend.Resulting lipid blend added to 25 mL propylene glycol (15 mg/mL). Study23 used low Ca⁺² lipid blend and Study 24 used lipid blend containingCa⁺² and Mg⁺² (370 and 54 μg/g, respectively). ^(k) Added 1 mL of astock 299 μg Ca⁺² per g PG, prior to addition of lipids

Consistent with the previous examples, these studies showedprecipitation occurred in the non-aqueous phospholipid solution whenhigh calcium or calcium and magnesium were present. This occurredregardless of if the calcium was present in the propylene glycol priorto the phospholipid addition, added after the phospholipid addition oradded with one of the components of the phospholipids (either withMPG5000 DPPE or in a phospholipid blend). Once the precipitate wasformed it did not disperse when mixed with aqueous solvent. Thisresulted in a cloudy aqueous preparation that had a reduced rate offiltration initially and often blocked the 0.2 μm filter (Table 5; FIG.5). The filtrate of cloudy aqueous preparations was dear butphospholipid measurement indicated consistently reduced levels of DPPA.This effect was apparent for both individually added phospholipids andphospholipids added as a blend.

Example 3.4 Effect of Non-Aqueous phospholipid Solution Addition toAqueous Solvent Containing Calcium

A series of studies were performed to examine the impact of calcium inthe aqueous solution on phospholipid suspension preparation. Theseinvolved the steps of: 1) preparing a non-aqueous phospholipid solution,2) preparing an aqueous solution and 3) combining solutions from 1 and2.

Example 3.4.1 Preparing Non-Aqueous Solution

Consistent with Example 1, the first step in Studies 26, 27 and 2.9 wereconducted as follows: DPPC, DPPA and MPEG5000-DPPE powder, characterizedas having low calcium concentration (see Table 1 in example methods),were added individually (in the sequence shown in Table 6) to heated(55° C.±5° C.) and stirred propylene glycol. It was verified by visualobservation that the phospholipid had fully dissolved and the resultingsolution was clear. These propylene glycol concentrates were transferredto the aqueous phase as described below.

Example 3.4.2 Preparing Aqueous Solution

In a separate vessel Sodium Chloride (NaCl), Sodium Phosphate DibasicHeptahydrate (Na₂HPO₄.7H₂O), and Sodium Phosphate Monobasic(NaH₂PO₄.H₂O) were added to water in a stirred vessel, and mixed untildissolved (for study 29 the Phosphate salts were excluded from theformulation). Propylene glycol and glycerol were also added as needed,so the final addition of non-aqueous phospholipid solution willreconstitute an 8:1:1 water:glycerol:propylene glycol composition. Insome studies, a solution of calcium acetate [Ca(OAc)₂] in water wasadded as indicated in Table 6. This aqueous solution was stirred,maintained at 55° C.±5° C. It was identified that the addition of 48.4μg/g calcium caused a marked flocculation in the aqueous solution in theabsence of any phospholipids (see Table 6, study A). At 12.2 μg/gcalcium no precipitation was produced in the aqueous solution (see Table6, study B).

Example 3.4.3 Combining Non-Aqueous and Aqueous Solutions

For all studies, the addition of the non-aqueous phospholipidconcentrate to the aqueous solution was done as follows: the warmphospholipid dissolved in propylene glycol was added and stirred at 100to 150 rpm. Visual observations were recorded and the time for fulldispersion or dissolution is stable (either clear or cloudy) noted. Forstudy 27, the aqueous formulation was initially clear. Calcium wastitrated and at concentrations ≥30.4 μg/g a cloudy precipitate wasformed (see Table 6). It was noted, however, that the aqueous solutionwithout phospholipid had a marked precipitated at 48.4 μg/g (Study A:Table 6). In study 27, at calcium levels where the aqueous solutionalone was not effected (12.2 μg/g based on study B, Table 6), no effectwas seen on aqueous Phospholipid formulation clarity. This was confirmedin study 26, where calcium was added to the aqueous solution (12.2 μg/g)prior to combining with the non-aqueous phospholipid concentrate. Thiswas further extended in study 29, where the phosphate buffer wasexcluded from the aqueous solution. Initially, calcium was added to theaqueous solution (12,2 μg/g) prior to combining with the non-aqueousphospholipid concentrate and the formulation was clear. Additionalcalcium was added after the phospholipid addition to the formulation upto 96 μg/g and no precipitation was observed.

The aqueous formulations from study 26 and 29 were then collected andfiltered through a 0.2 μm filter at 55° C. under 5 psi head pressure.Flow rate at 10 minutes was not reduced compared to initial flow; allthe sample was filtered and overall filtration was similar topreparations not containing calcium (see studies 19, 2.3 and 25). Pre-and post-filtration samples were collected and compared to determineloss of phospholipids associated with filtration. No meaningful. loss ofPhospholipid was apparent (see Table 6).

TABLE 6 Effect of non-aqueous phospholipid solution addition to aqueoussolvent containing divalent metal ions Aqueous suspension ^(a)Non-aqueous Appearance Percent of lipid concentrate Calcium after lipidInitial % phospholipid Phospholipid concentration concentrate ContainsFiltration post filtration ^(d) addition Observed in aqueous addition toPO₄ Rate at 9-10 MPEG5000- Study to PG^(b) cloudiness ^(c) (μg Ca⁺²/g)aqueous buffer minutes DPPC DPPA DPPE 27 C, E, A 0 Titration Clear to12.2, Yes n/a 0 to 48.7 μg slightly cloudy Ca+²/g at 30.4 and cloudywith precipitate at 36.5 μg Ca⁺²/g water 26 C, E, A 0 12.2 ^(e) ClearYes 114.3 99 101 98 29 C, A, E 0 12.2 ^(e) Clear No 100.8 99  99 98 An/a n/a 48.4 ^(e) Precipitate Yes n/a n/a n/a n/a B n/a n/a 12.2 ^(e)Clear Yes n/a n/a n/a n/a ^(a) All compounding performed at 55° C.Non-aqueous phospholipid solutions were added to aqueous compoundingvessel containing: water (800 mg); sodium phosphate heptahydrate (2.16mg/mL), sodium phosphate monohydrate (2.34 mg/mL), sodium chloride (4.84mg/mL), glycerol (126 mg), and propylene glycol (51.75 mg) unlessotherwise indicated in footnotes. ^(b)“A” is DPPA (0.9 mg/mL), “C” isDPPC (8.02 mg/mL) and “E” is MPEG5000-DPPE (6.08 mg/mL) which were addedin the order listed, to 25 mL propylene glycol ^(c) Defined in methodsSection 1.4 ^(d) HPLC with CAD detection described in Section 1.6 ^(e)Prior to addition of lipid concentrate to aqueous compounding vessel, 1mL, 1 mL, 4 mL and 1 mL of calcium acetate concentrate (6.085 mg Ca⁺²per g of water) was added for studies 26, 29, A and B, respectively.

These studies demonstrate that calcium is not causing phospholipidprecipitation in the aqueous formulation even up to 96 μg/g. However, atcalcium levels higher than 12.2 μg/g, the phosphate salts start toprecipitate.

Example 4 Effect of Divalent Metal Ions on phospholipid Dissolving inBuffered propylene glycol and with the Addition of glycerol Example 4.1Calcium Titration in Buffered Non-Aqueous phospholipid Concentrate

Studies 31 and 32 were conducted as follows: DPPC, DPPA andMPEG5000-DPPE powder, characterized as having low calcium concentration(see Table 1 in example methods), were added either individually (in thesequence shown in Table 7) or as a phospholipid blend (made usingtoluene and methanol to dissolve and adding MTBE to precipitate out thelipid blend) to heated (55° C.±5° C.) and stirred acetate bufferedpropylene glycol. It was verified by visual observation that the lipidhad fully dissolved and the resulting solution was clear (see examplemethods Section 1.4). A solution of calcium acetate [Ca(OAc)₂] inpropylene glycol was used to titrate the phospholipid solution by aseries of small additions. The solution was stirred and observed forchanges in appearance during the titration, as compared. to a solventblank after each addition and the assessment of clarity was recorded. Acloudiness score (see Section 1.4, FIG. 1, for method) based on thisassessment was made and the lowest calcium concentration producing a +,++, and +++ score is shown in Table 7.

TABLE 7 Effect of calcium on phospholipid dissolving in bufferedpropylene glycol Order of lipid addition ^(a) Calcium Observedcloudiness MPEG5000- Lipid concentration thresholds (μg/mL Ca⁺²) ^(b)Study DPPC DPPA DPPE blend (μg/mL Ca+2) + ++ +++ 31 n/a n/a n/a 1^(c)Titration ^(r) 5.8 11.2 22.3 32 1 3 2 n/a Titration ^(s) 11.3 17.0 >33.5^(a) Individual lipids [DPPC (4.01 mg), DPPA (0.45 mg), andMPEG5000-DPPE (3.04 mg), in the order listed] or lipid blend (7.5 mg)were added to each mL of propylene glycol containing sodium acetate(0.74 mg) and acetic acid (0.06 mg), at 60° C. with stirring. ^(b)Defined in methods Section 1.4 ^(c) Made using toluene and methanol todissolve lipids and adding MTBE to precipitate out the lipid blend.

Example 4.2 Calcium Titration in Buffered Non-Aqueous phospholipidConcentrate from MPEG5000-DPPE

Studies 33 through 36 were conducted as follows: DPPC, DPPA and eitherhigh (980 ppm, Lot 1 or low Ca⁺² (4 ppm) containing MPEG5000-DPPE, wereadded individually (in the sequence shown in Table 8) or as aphospholipid blend (made using toluene and methanol to dissolve andadding MTBE to precipitate out the lipid blend) to heated (55° C.±5° C.)and stirred. acetate buffered propylene glycol. Clarity was assessed(see example methods Section 1.4) and cloudiness was observed (+ or ++;see example methods, FIG. 1) with the phospholipid blend containing highcalcium. This contrasted with the clear solution produced by dissolvinglow calcium containing phospholipid blend (see Table 8).

Example 4.3 Glycerol Addition

To these buffered non-aqueous phospholipid solutions, glycerol wastransferred with stirring at 300 rpm. Many gas bubbles were trapped inthe mixing solution but cleared once the stirrer was stopped. Visualobservations were recorded and the clarity level (either clear orcloudy) noted. These PG/G solutions were then collected and filteredthrough a 0.2 μm filter at 60° C. under 10 psi head pressure. Flow ratewas measured and samples collected for phospholipid measurement. Pre-and post-filtration samples were compared to determine loss ofphospholipids associated with filtration.

TABLE 8 Effect of Calcium on phospholipid dissolving in bufferedpropylene glycol and glycerol added Propylene glycol with added glycerol^(b) Acetate/Propylene Percent glycol concentrate ^(a) Ca⁺² and (Mg⁺²)of Initial % phospholipid Lipid Appearance concentration Filtration postfiltration ^(d) addition Ca⁺² (Mg⁺²) Observed after addition productRate at 8-9 MPEG 5000 Study to PG [μg/g] cloudiness ^(c) of glycerol[μg/g] minute DPPC DPPA DPPE 33 LB ^(e) 0 (0) 0 clear 0 174.2 97 90 9634 LB ^(e) 2.7 (0.4) + cloudy 1.6 (0.2) 96.7; 101 86 98 blocked filter35 C, A, E 0 (0) 0 clear 0 245.2 97 100 98 36 C, A, E^(f) 2.9 (0.4) ++cloudy 1.7 (0.2) 19.8: 99 80 100 blocked filter ^(a) Individual lipids[DPPC (4.01 mg), DPPA (0.45 mg), and MPEG5000-DPPE (3.04 mg), in theorder listed] or lipid blend (7.5 mg) were added to each mL of propyleneglycol containing sodium acetate (0.74 mg) and acetic acid (0.06 mg), at60° C. with stirring. ^(b) Propylene glycol containing acetate bufferand phospholipids is diluted 1:1 (v/v) with glycerol. ^(c) Defined inmethods Section 1.4 ^(d) HPLC with CAD detection described in Section1.6 ^(e) Lipid blend made using toluene and methanol to dissolve lipidsand adding MTBE to precipitate out the lipid blend, low Ca+2 (Lot 1) forstudy 33, and high Ca+2 (370 Ca⁺²and 54 ppm Mg⁺²; Lot 2) for Study 34.^(f)MPEG5000-DPPE containing Ca⁺² (3.04 mg/mL; 980 and 150 ppm Ca⁺² andMg⁺²; Lot 1) used in this experiment

These studies showed precipitation occurred in the buffered non-aqueousphospholipid solution in a calcium concentration dependent manner. Thisoccurred regardless of whether the buffered non-aqueous phospholipidsolution was made with individual phospholipids or a lipid blend and atconcentrations that were not meaningfully different. The concentrationto cause initial precipitation for the buffered solution was higher (5.8to 11.3 Ca⁺²) than for the non-buffered solutions (1.5-2.3 μg Ca⁺²/g:see Table 2, studies 1 through 4) indicating an influence of the buffer.

Calcium front the lipid blend caused precipitation in the bufferednon-aqueous phospholipid solution as was seen in the non-bufferedsolution. Once the precipitate was formed it did not disperse when mixedwith glycerol. This results in a cloudy non-aqueous formulation that hada reduced rate of filtration initially and often blocked the 0.2 μmfilter (Table 8, FIG. 6). The filtrate of cloudy preparations was clearbut phospholipid measurement indicated slightly reduced levels of DPPA.

Example 5 Microsphere Formation and Acoustic Detection of ManufacturedProduct Example 5.1 Aqueous phospholipid Suspension

Studies 37 and 38 were conducted as follows: filtered materials fromstudy 19 and 23 were prepared in vials (see examples method Section1.7.1). Following VIALMIX®, activation samples were analyzed formicrosphere size and number (see methods Section 1.7.3) and clinicalultrasound acoustic attributes (see methods Section 1.7.4), see Table 9.

TABLE 9 Aqueous phospholipid suspension Microsphere number and Size andacoustic activity. Mean Microsphere Acoustic Mean Microsphere (SD)Diameter per mL Attenuation^(c) (microns)^(a) (×10⁹)^(b) (dB/cm/10⁶Study Production basis N = 2 N = 2 bubbles/mL) 37 Individualphospholipids with low 1.38, 1.36 3.73, 2.92 8.9 (0.3) Ca⁺² measured inMPEG-5000 DPPE and other components 38 Phospholipid blend with low 1.34,1.35 3.4, 2.5 9.0 (1.3) Ca⁺² measured in MPEG-5000 DPPE and othercomponents ^(a)Mean microsphere diameter for microspheres ranging from 1to 80 microns. ^(b)Mean microsphere concentration for microspheresranging from 1 to 80 microns. ^(c)see example methods section fordetails

These studies demonstrate an aqueous phospholipid suspension can beproduced using individual phospholipids or a phospholipid blend when thecomponents have a low calcium concentration. Both products havemicrosphere diameter within the specification of DEFINITY® (seeDEFINITY® package insert) and have strong ultrasound acousticattenuation on a clinical ultrasound machine.

Aspects and Embodiments

Various aspects and embodiments provided by this disclosure are listedbelow.

Clause 1. A method for preparing a phospholipid suspension, comprising

providing DPPA, DPPC and MREG5000-DPPE stocks,

measuring calcium concentration of one or more of the DPPC, DPPA andMPEG5000-DPPE stocks,

combining the DPPA, DPPC and/or MPEG5000-DPMI; stocks with a non-aqueoussolvent to form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 2. The method of clause 1, further comprising measuring calciumconcentration of the non-aqueous solvent.

Clause 3. The method of clause 1, wherein the combined measured calciumconcentration of the DPPA, DPPC and/or MPEG-DPPE stocks is low.

Clause 4. The method of clause 1 or 3, wherein the combined measuredcalcium concentration of the DPPA, DPPC and/or MPEG-DPPE stocks and thenon-aqueous solvent is low.

Clause 5. The method of clause 1, wherein the calcium concentrations ofthe DPPC, DPPA and MPEG5000-DPPE stocks are measured.

Clause 6. The method of clause 2, wherein the calcium concentrations ofthe DPPC, DPPA and MPEG5000-DPPE stocks are measured and the combinedmeasured calcium concentration of the DPPA, DPPC, MPEG-DPPE stocks andthe non-aqueous solvent is low.

Clause 7. A method for preparing a phospholipid suspension, comprising

providing DPPA, DPPC and MPEG5000-DPPE stocks,

measuring calcium concentration of one or more of the DPPC, DPPA1PEC15000-DPPE stocks,

combining DPPA, DPPC and/or MPEG5000-DPPE stocks having a combinedmeasured low calcium concentration with a non-aqueous solvent to form aphospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 8. The method of clause 7, wherein the calcium concentration ofthe non-aqueous solvent is measured and the DPPA, DPPC, MPEG500-DPPEstocks and the non-aqueous solvent have a combined measured low calciumconcentration.

Clause 9. A method for preparing a phospholipid suspension, comprising

combining a MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and anon-aqueous solvent, each with a characterized calcium concentration toform a phospholipid solution, wherein the combined characterized calciumconcentration of the IMPEG5000-DPPE stock, the DPPA stock, the DPPCstock and the non-aqueous solvent is a low calcium concentration, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 10. A method for preparing a phospholipid suspension, comprising

selecting a MPEG5000-DPPE stock, a DPPA stock and a DPPC stock, one, twoor all three of which have a characterized calcium concentration,wherein the combined characterized calcium concentration is a lowcalcium concentration,

combining said MPEG5000-DPPE stock, DPPA stock, DPPC stock and anon-aqueous solvent to form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent form aphospholipid suspension.

Clause 11. A method for preparing a phospholipid suspension, comprising

selecting a MPEG5000-DPPE stock, a DPPA stock and a DPPC stock, eachwith characterized calcium concentration, wherein the combinedcharacterized calcium concentration is a low calcium concentration,

combining said MPEG5000-DPPE stock, DPPA stock. DPPC stock and anon-aqueous solvent to form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 12. The method of clause 11 wherein the non-aqueous solvent has acharacterized calcium concentration, and the combined characterizedcalcium concentration of the MPEG5000-DPPE, DPPA and DPPC stocks and thenon-aqueous solvent is low.

Clause 13. A method for preparing a phospholipid suspension, comprising

measuring calcium concentration of a MPEG5000-DPPE stock,

combining a MPEG5000-DPPE stock having a measured low calciumconcentration with a DPPA stock, a DPPC stock, and a non-aqueous solventto form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 14. The method of clause 11, wherein the non-aqueous solventcomprises (i) propylene glycol or (ii) propylene glycol and glycerol.

Clause 15. The method of clause 13 or 14, wherein the non-aqueoussolvent comprises a buffer.

Clause 16. The method of clause 13 or 1.4, wherein the non-aqueoussolvent comprises an acetate buffer.

Clause 17. The method of clause 13 or 14, wherein the aqueous solventcomprises a buffer.

Clause 18. The method of clause 13 or lit, wherein the aqueous solventcomprises a phosphate buffer.

Clause 19. The method of any one of clauses 13-18, wherein the DPPC,DPPA and MPEG5000-DPPE stocks are individually combined with thenon-aqueous solvent to form the phospholipid solution.

Clause 20. The method of any one of clauses 13-18, wherein the DPPC'DPPA and MPEG5000-DPPE stocks are sequentially combined with thenon-aqueous solvent, in an order-independent manner, to form thephospholipid solution.

Clause 21. The method of any one of clauses 13-18, wherein the DPPC,DPPA and MPEG5000-DPPE stocks are combined with each other to form aphospholipid mixture and the phospholipid mixture is then combined withthe non-aqueous solvent to form the phospholipid solution.

Clause 22. The method of any one of clauses 13-18, wherein the DPPC,DPPA and MPEG5000-DPPE stocks are combined with each other to form aphospholipid blend, and the phospholipid blend is combined with thenon-aqueous solvent to form the phospholipid solution.

Clause 23. The method of clause 22, wherein the phospholipid blend isformed using an organic solvent dissolution-precipitation processcomprising dissolving the DPPC, DPPA and MPEG5000-DPPE stocks into amixture of methanol and toluene, optionally-concentrating thephospholipid/methanol/toluene mixture, and then contacting theconcentrated phospholipid/methanol/toluene mixture with methyl t-butylether (MTBE) to precipitate the phospholipids to form the phospholipidblend.

Clause 24. The method of any one of clauses 13-23, wherein the lowcalcium concentration is less than 115 ppm.

Clause 25. The method of any one of clauses 13-24, further comprisingplacing the phospholipid suspension in a vial and introducing aperfluorocarbon gas into the headspace of the vial.

Clause 26. The method of clause 25, further comprising activating thephospholipid suspension with the perfluorocarbon gas to form anultrasound contrast agent comprising phospholipid-encapsulated gasmicrospheres.

Clause 27. The method of clause 26, further comp! administering theultrasound contrast agent to a subject and obtaining one or morecontrast-enhanced ultrasound images of the subject.

Clause 28. The method of any one of clauses 13-27, further comprisingmeasuring calcium concentration of the DPPA stock and/or DPPC stockand/or phospholipid mixture and/or phospholipid blend.

Clause 29. A method for preparing a phospholipid suspension, comprising

measuring calcium concentration of a DPPC stock,

combining a DPPC stock having a measured low calcium concentration witha DPPA stock, a MPEC15000-DPPE stock, and a non-aqueous solvent to forma phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 30. The method of clause 2 wherein the low calcium concentrationis less than 90 ppm.

Clause 31. A method for preparing a phospholipid suspension, comprising

measuring calcium concentration of a DPPA stock,

combining a DPPA stock having a measured low calcium concentration witha DPPC stock, a MPEG5000-DPPE stock, and a non-aqueous solvent to form aphospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 32. The methodof clause 31, wherein the low calcium concentrationis less than 780 ppm.

Clause 33. A method for preparing a phospholipid suspension, comprising

measuring calcium concentration of a non-aqueous solvent,

combining a non-aqueous solvent having a measured low calciumconcentration with a DPPA stock, a DPPC stock, and a MPEC35000-13PPEstock, to form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 34. The method of clause 33, wherein the low calciumconcentration is less than 0.7 ppm.

Clause 35. A method for preparing a phospholipid suspension, comprising

selecting a MPEG5000-DPPE stock characterized as having no or lowcalcium concentration,

combining said MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and anon-aqueous solvent to form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 36. The method of clause 35, wherein the MPEG5000-I PPE stock isfurther characterized as having no or low divalent metal cation content.

Clause 37. A method for preparing a phospholipid suspension, comprising

combining a MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and anon-aqueous solvent to form a phospholipid solution characterized ashaving no or low calcium concentration, and

combining the phospholipid solution with an aqueous solvent o form aphospholipid suspension.

Clause 38. A method for imaging a subject comprising

combining a phospholipid suspension with a perfluorocarbon gas to forman ultrasound contrast agent comprising phospholipid-encapsulated gasmicrospheres,

administering the ultrasound contrast agent to a subject, and

obtaining one or more contrast-enhanced ultrasound contrast images ofthe subject, wherein the phospholipid suspension is prepared by themethod of any one of clauses 1-37.

Clause 39. A method for imaging a subject comprising

combining a phospholipid suspension with a perfluorocarbon gas to forman ultrasound contrast agent comprising phospholipid-encapsulated gasmicrospheres,

administering the ultrasound contrast agent to a subject, and

obtaining one or more contrast-enhanced ultrasound contrast images ofthe subject, wherein the phospholipid suspension is prepared by a methodcomprising

measuring calcium concentration of MPEG5000-DPPE stock,

combining a MPEG5000-DPPE stock having a measured low calciumconcentration with a DPPA stock, a DPPC stock, and a non-aqueous solventto form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form thephospholipid suspension.

Clause 40. A method for imaging a subject comprising

combining a phospholipid suspension with a perfluorocarbon gas to forman ultrasound contrast agent comprising phospholipid-encapsulated gasmicrospheres,

administering the ultrasound contrast agent to a subject, and

obtaining one or more contrast-enhanced ultrasound contrast images ofthe subject, wherein the phospholipid suspension is prepared by a methodcomprising

selecting a MPEG5000-DPPE stock characterized as having no or lowcalcium concentration,

combining said MPEG5000-DPPE stock a DPPA stock, a DPPC stock and anon-aqueous solvent to form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 41. A method for imaging a subject comprising

combining a phospholipid suspension with a perfluorocarbon gas to forman ultrasound contrast agent comprising phospholipid-encapsulated gasmicrospheres,

administering the ultrasound contrast agent to a subject, and

obtaining one or more contrast-enhanced ultrasound contrast images ofthe subject, wherein the phospholipid suspension is prepared by a methodcomprising

combining a MPEG5000-DPPE stock, a DPPA stock, a DPPC stock and anon-aqueous solvent to form a phospholipid solution characterized ashaving no or low calcium concentration, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 42. A method for preparing a phospholipid suspension, comprising

individually combining DPPA, DPPC and MPEG5000-DPPE stocks with apropylene glycol (PG) -comprising non-aqueous solvent, in a low or nocalcium condition, to form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 43. A method for preparing a phospholipid suspension, comprising

sequentially combining DPPA, DPPC and MPEG5000-DPPE stocks with aPG-comprising non-aqueous solvent, in a low or no calcium condition, inan order-independent manner, to form a phospholipid solution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 44. A method for preparing a phospholipid suspension, comprising

combining in a methanol and toluene-free condition, DPPA, DPPC andMPEG5000-DPPE stocks to form a phospholipid blend,

combining the phospholipid blend with a PG-comprising non-aqueoussolvent, in a low or no calcium condition, to form a phospholipidsolution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 45. A method for preparing a phospholipid suspension, comprising

combining DPPA, DPPC and MPEG5000-DPPE stocks with a blend solvent toform a phospholipid blend,

evaporating the blend solvent to form a dried phospholipid blend,

combining the dried phospholipid blend with a PG-comprising non-aqueoussolvent, in a low or no calcium condition, to form a phospholipidsolution, and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 46. A method for preparing a phospholipid suspension, comprising

combining DPPA, DPPC and MPEG5000-DPPE stocks with a blend solvent toform a phospholipid blend,

precipitating, in a MTBE-free condition, the phospholipid blend using asecond blend solvent,

combining the precipitated phospholipid blend with a non-aqueoussolvent, in a low or no calcium condition, to form a phospholipidsolution and

combining the phospholipid solution with an aqueous solvent to form aphospholipid suspension.

Clause 47. The method of any one of clauses 42-4 wherein the no or lowcalcium concentration is less than 0.7 ppm.

Clause 48. The method of any one of clauses 42-47, further comprisingcombining the phospholipid suspension with a perfluorocarbon gas to forman ultrasound contrast agent comprising phospholipid-encapsulated gasmicrospheres.

Clause 49. The method of clause 48, further comprising administering theultrasound contrast agent to a subject and obtaining one or morecontrast-enhanced ultrasound images of the subject.

Clause 50. A method for imaging a subject comprising

combining a phospholipid suspension with a perfluorocarbon gas to forman ultrasound contrast agent comprising phospholipid-encapsulated gasmicrospheres,

administering the ultrasound contrast agent to a subject, and

obtaining one or more contrast-enhanced ultrasound contrast images ofthe subject, wherein the phospholipid suspension is prepared by themethod of any one of clauses 42-47.

Clause 51. A composition comprising

a phospholipid solution comprising DPPA, DPPC and MPEG5000-DPPE in anon-aqueous solvent and having a low calcium concentration.

Clause 52. A composition comprising

a phospholipid solution comprising DPPA, DPPC and MPEG5000-DPPE in anon-aqueous solvent, wherein the DPPA, DPPC and MPEG5000-DPPE and thenon-aqueous solvent have a combined characterized calcium ion contentthat is low.

Clause 53. The composition of clause 51 or 52, wherein the non-aqueoussolvent comprises propylene glycol.

Clause 54. The composition of clause 51 or 52, wherein the non-aqueoussolvent comprises propylene glycol and glycerol.

Clause 55. The composition of any one of clause 51-54, wherein thenon-aqueous solvent comprises a buffer.

Clause 56. The composition of clause 55, wherein the buffer is acetatebuffer.

Clause 57. The composition of any one of clauses 51-56, furthercomprising a perfluorocarbon gas.

Clause 58. The composition of clause 57, therein the perfluorocarbon gasis perflutren.

Clause 59. A method of ultrasound contrast imaging a subject comprising

(a) activating a phospholipid suspension with a perfluorocarbon gas toform lipid-encapsulated gas microspheres, wherein the phospholipidsuspension comprises a phospholipid solution having one or morephospholipids and a non-aqueous solvent, one or more of which has acharacterized low calcium concentration,

(b) administering the lipid-encapsulated gas microspheres to a subject,and

(c) obtaining an ultrasound image of the subject.

Clause 60. The method of clause 59, wherein the one or morephospholipids comprise DPPC and MPEG-5000-DPPE.

Clause 61. The method of clause 59. wherein the one or morephospholipids comprise DPPA, DPPC and MPEG-5000-DPPE.

Clause 62. The method of clause 61, wherein DPPA, DPPC and 1PEG5000-DPPEare present in a mole % ratio of 10 to 82 to 8 (10:82:8).

Clause 63. The method of any one of clauses 60-62, wherein thecharacterized low calcium concentration for DPPA is less than 780 ppm,for DPPC is less than 90 ppm, and for MPEG5000-DPPE is less than 115ppm.

Clause 64. The method of any one of clauses 59-63, wherein thenon-aqueous solvent comprises (a) propylene glycol, or (b) propyleneglycol and glycerol.

Clause 65. The method of any one of clauses 59-64, wherein thecharacterized low calcium concentration for the non-aqueous solvent isless than 0.7 ppm.

Clause 66. The method of any one of clauses 59-65, wherein thephospholipid solution has no detectable phospholipid precipitate.

Clause 67. A method of ultrasound contrast imaging a subject comprising

(a) activating a phospholipid suspension with a perfluorocarbon gas toform lipid-encapsulated gas microspheres, wherein the phospholipidsuspension comprises a phospholipid solution having one or morephospholipids and a non-aqueous solvent and made under a methanol andtoluene free condition and a methyl t-butyl ether free condition,wherein one or more of the phospholipids and non-aqueous solvent has alow calcium concentration,

(b) administering the lipid-encapsulated gas rnicrospheres to a subject,and

(c) obtaining an ultrasound image of the subject.

Clause 68. The method of clause 67, wherein the one or morephospholipids comprise DPPC and MPEG-5000-DPPE.

Clause 69. The method of clause 67, wherein the one or morephospholipids comprise DPPA, DPPC and MPEG-5000-DPPE.

Clause 70. The method of clause 69, wherein DPPA, DPPC andMPEG-5000-DPPE are present in a mole % ratio of 10 to 82 to 8 (10:82:8).

Clause 71. The method of any one of clauses 67-70, wherein the lowcalcium concentration for DPPA is less than 780 ppm, for DPPC is lessthan 90 ppm, and for MPEG5000-DPPE is less than 115 ppm.

Clause 72. The method of any one of clauses 67-71, wherein thenon-aqueous solvent comprises (a) propylene glycol, or (h) propyleneglycol and glycerol.

Clause 73. The method of any one of clauses 67-72, wherein the lowcalcium concentration for the non-aqueous solvent is less than 0.7 ppm.

Clause 74. The method of any one of clauses 67-73, therein thephospholipid solution has no detectable phospholipid precipitate.

Clause 75. A method for preparing lipid-encapsulated gas microspherescomprising

combining one or more phospholipids and a non-aqueous solvent to form aphospholipid solution, wherein one or more of the phospholipids and/orthe non-aqueous solvent, has a characterized low calcium concentration,

combining the phospholipid solution with an aqueous solution to form aphospholipid. suspension, and

activating the phospholipid suspension with a perfluorocarbon gas toform lipid-encapsulated gas microspheres.

Clause 76. The method of clause 75, wherein the one or morephospholipids comprise DPPC and MPEG-5000-DPPF.

Clause 77. The method of clause 75, wherein the one or morephospholipids comprise DPPA, DPPC and MPEG-5000-DPPE.

Clause 78. The method of clause 77, wherein DPPA, DPPC andMPEG-5000-DPPE are present in a mole % ratio of 10 to 82 to 8 (10:82:8).

Clause 79. The method of any one of clauses 75-78, wherein thecharacterized low calcium concentration for DPPA is less than 780 ppm,for DPPC is less than 90 ppm, and for MPEG5000-DPPE is less than 115ppm.

Clause 80. The method of any one of clauses 75-79, wherein thenon-aqueous solvent comprises (a) propylene glycol or (b) propyleneglycol and glycerol.

Clause 81. The method of any one of clauses 75-80, wherein thecharacterized low calcium concentration for the non-aqueous solvent isless than 0.7 ppm.

Clause 82. The method of any one of clauses 75-81, -herein thephospholipid solution has no detectable phospholipid precipitate.

Clause 83. A method for preparing lipid-encapsulated gas microspherescomprising

combining one or more phospholipids and a non-aqueous solvent, in amethanol and toluene free and methyl t-butyl ether free condition, toform a phospholipid solution, wherein one or more of the phospholipidsand/or the non-aqueous solvent has a low calcium concentration.

combining the phospholipid solution with an aqueous solution to form aphospholipid suspension, and

activating the phospholipid suspension with a perfluorocarbon gas, toform lipid-encapsulated gas microspheres.

Clause 84. The method of clause 83, wherein the one or more lipidscomprise (a) DPPC and MPEG-5000-DPPE, or (b) DPPA, DPPC andMPEG-5000-DPPE and/or (c) DPPA, DPPC and MPEG-5000-DPPE in a mole %ratio of 10 to 82 to 8 (10:82:8).

Clause 85. The method of clause 84, wherein the low calciumconcentration for DPPA is less than 780 ppm, for DPPC is less than 90ppm, and for MPEG5000-DPPE is less than 115 ppm.

Clause 86. The method of any one of clauses 83-85, wherein thenon-aqueous solvent comprises (a) propylene glycol, or (b) propyleneglycol and glycerol.

Clause 87. The method of any one of clauses 83-86, wherein the lowcalcium concentration for the non-aqueous solvent is less than 0.7 ppm.

Clause 88. The method of any one of clauses 83-87, wherein thephospholipid solution has no detectable phospholipid precipitate.

Clause 89. The method of any one of clauses 67-74, wherein one or moreof the phospholipids or the non-aqueous solvent has a characterized lowcalcium concentration.

Clause 90. The method of clause 89, wherein the characterized lowcalcium concentration is determined using atomic absorptionspectroscopy.

Clause 91. The method of any one of clauses 67-74, wherein the o calciumconcentration is determined using atomic absorption spectroscopy.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in sonic cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary. should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e.. the inclusion of at least one, butalso including, more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other titan the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one. A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods chinned herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatient Examining Procedures, Section 2111.03.

1-27. (canceled)
 28. A method of ultrasound contrast imaging a subject comprising (a) combining one or more phospholipids and a non-aqueous solvent, in the presence of one or two but not all three of methanol, toluene and methyl t-butyl ether (MTBE), to form a phospholipid solution having calcium and/or magnesium divalent metal cation present at a total divalent metal cation concentration of less than 0.7 parts per million (ppm), and (b) combining the phospholipid solution with an aqueous solution to form a phospholipid suspension, and (c) activating the phospholipid suspension with a perfluorocarbon gas to form lipid-encapsulated gas microspheres, (d) administering the lipid-encapsulated gas microspheres to a subject, and (e) obtaining an ultrasound image of the subject.
 29. The method of claim 28, wherein the one or more phospholipids comprise (a) DPPC and MPEG-5000-DPPE or (b) DPPA, DPPC and MPEG-5000-DPPE.
 30. The method of claim 28, wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE.
 31. The method of claim 29, wherein DPPC has a combined calcium and magnesium concentration of less than 90 ppm, and MPEG5000-DPPE has a combined calcium and magnesium concentration of less than 115 ppm.
 32. The method of claim 28, wherein the non-aqueous solvent comprises propylene glycol.
 33. The method of claim 28, wherein the non-aqueous solvent has a combined calcium and magnesium concentration of less than 0.7 ppm.
 34. The method of claim 28, wherein the non-aqueous solvent comprises propylene glycol and glycerol.
 35. The method of claim 28, wherein the phospholipid solution has no detectable phospholipid precipitate.
 36. The method of claim 28, wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE in molar percentages of 5-15%, 77-90% and 5-15%, respectively.
 37. The method of claim 28, wherein the one or more phospholipids comprise DPPA, DPPC and MPEG-5000-DPPE in a mole % ratio of 10 to 82 to 8 (10:82:8).
 38. The method of claim 28, wherein the perfluorocarbon gas is perfluoropropane gas.
 39. The method of claim 28, wherein the one or more phospholipids comprise DPPC and MPEG-5000-DPPE, the phospholipid solution comprises DPPC, MPEG5000-DPPE and propylene glycol, the aqueous solvent comprises water, propylene glycol and glycerol, and the phospholipid suspension comprises DPPC, MPEG5000-DPPE, propylene glycol, water and glycerol.
 40. The method of claim 28, wherein the aqueous solvent further comprises one or more salts.
 41. The method of claim 28, wherein the aqueous solvent further comprises one or more buffers.
 42. The method of claim 29, wherein the MPEG5000-DPPE has a combined calcium and magnesium concentration of less than 115 ppm.
 43. The method of claim 28, wherein the phospholipid solution is formed in the presence of methanol.
 44. The method of claim 29, wherein the phospholipid solution is formed in the presence of methanol.
 45. The method of claim 30, wherein the phospholipid solution is formed in the presence of methanol.
 46. The method of claim 28, wherein the phospholipid solution is formed in the presence of toluene.
 47. The method of claim 29, wherein the phospholipid solution is formed in the presence of toluene.
 48. The method of claim 30, wherein the phospholipid solution is formed in the presence of toluene.
 49. The method of claim 28, wherein the phospholipid solution is formed in the presence of methanol and toluene.
 50. The method of claim 29, wherein the phospholipid solution is formed in the presence of methanol and toluene.
 51. The method of claim 30, wherein the phospholipid solution is formed in the presence of methanol and toluene.
 52. The method of claim 28, wherein the phospholipid solution is formed in the presence of MTBE.
 53. The method of claim 29, wherein the phospholipid solution is formed in the presence of MTBE.
 54. The method of claim 30, wherein the phospholipid solution is formed in the presence of MTBE. 